key: cord-0972152-ecilsjts
authors: Raghav, S. K.; Sen, K.; Ghosh, A.; Datta, S.; Ahad, A.; Jha, A.; Chatterjee, S.; Suranjika, S.; Sengupta, S.; Bhattacharya, G.; Shriwas, O.; Avula, K.; Kshatri, J.; Prasad, P.; Parida, A. K.
title: SARS-CoV-2 specific immune-signature in direct contacts of COVID-19 cases protect them from contracting disease: A Retrospective Study
date: 2021-03-12
journal: nan
DOI: 10.1101/2021.03.11.21253367
sha: e5912a59c5ba67c29f7d70efcd075d6e1c4929c0
doc_id: 972152
cord_uid: ecilsjts

The response to SARS-CoV-2 is largely impacted by the level of exposure and the status of immunity. The nature of protection shown by direct contacts of COVID-19 positive patients is quite intriguing to note. We aimed to study the immune differences reinforcing contact individuals in circumventing the disease. Our observation showed direct contacts of PCR positive patients developed elevated neutralizing antibody titres and cytokine levels. On the other hand, single cell data revealed differential usage of V(D)J genes and unique BCR clonotypes imparting protective immune signatures.

Off late the incidence of new cases of COVID-19 has declined remarkably in certain regions which has led to the plausible explanation of hygiene hypothesis or prevalence of antibodies with neutralizing capabilities [1, 2, 3] . The humoral immune response plays a pivotal role in evading viral infections. Several reports have appreciated the role of serum IgA for early neutralizing response against SARS-CoV-2 and their longevity for months after onset of symptoms [4, 5, 6, 7] . The induction of humoral immune response can increase the efficacy of vaccination [8] . Last year during the period of June to July large groups of migrant workers started returning back to their native residence in shared transports. From the SARS-CoV-2 RT-qPCR surveillance information of the migrant workers we observed that some individuals have not contracted the disease even after travelling in close vicinity (proximity <1m) of COVID-19 bearing individuals. To understand the immune status of these individuals (direct contacts <1m proximity) we enrolled three subject groups for our study namely control (CTRL, unexposed to SARS-CoV-2), infected (INF, COVID-19 positive, inception confirmed by RT-qPCR test) and contact (CON, individuals who travelled with SARS-CoV-2 RT-qPCR positive individuals in the same vehicle sitting within ~1m radius for 3-4 days). We further divided the infected individuals into two subgroups viz symptomatic (SYM, showing mild or moderate symptoms) and asymptomatic (ASY) based on disease severity at the time of sample collection. This study has taken into account candidates of both sexes with age ranging from 4 to 60 years.

. 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 tests were performed according to the manufacturer's protocol.

Human cytokine quantification was performed from serum samples according to manufacturer's protocol (Bio-Plex Pro Human Cytokine Screening Panel #10000092045, Part no-12007283).

Briefly, 50 µl of 1x beads were added to the wells and washed 2x times with 200 µl of wash buffer.

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The copyright holder for this preprint this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253367 doi: medRxiv preprint 5 50 µl of standards, samples (1:4 diluted) and controls were added and incubated on the shaker at 850 rpm for 30 minutes at RT. Following which in the same manner 25 µl of 1x detection antibody was mixed and incubated. Thereafter, the wells were washed and streptavidin-PE was added for 10 minutes with shaking. Eventually after giving final washes, the samples were resuspended in 125 µl of assay buffer and data acquisition was performed on Bio-Plex 200 System. 

10X Chromium platform BCR amplification and library preparation kits (10X Genomics) were used for preparing the libraries. Next generation sequencing was performed using Illumina NextSeq 550 platform. The BCR sequences for each single cell were assembled by the Cell Ranger pipeline (v5.0.0). After identification of the CDR3 sequences and the rearranged BCR genes, analysis was . 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 March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253367 doi: medRxiv preprint 6 performed using Loupe V(D)J Browser v.2.0.1. BCR diversity metric, containing clonotype frequency and barcode information was processed using Seurat (v3) and plotted using GGPLOT2 R package.

Neutralization assay was performed according to manufacturer's protocol (SARS-CoV-2 Surrogate Virus Neutralization Test Kit, Genscript # L00847-A). Briefly, positive control, negative control and samples (serum 1:4 dilution) were diluted with HRP-RBD in a ratio of 1:1 in tubes and incubated at 37 for 30 minutes. After incubation 100 µl of each mixture were added to the plate for 15 minutes at 37 . Following which the wells were washed with 1x wash solution 4x times and finally TMB substrate was added for development of colour. The reaction was stopped using 50 µL of stop solution and absorbance was measured immediately in a Multiskan reader (Thermo scientific). The percent inhibition/neutralization was calculated using the formula = (1 -OD value of Sample /OD value of Negative Control) × 100%.

All the statistical tests are performed in R and the bar and box plots are generated using the GGPUBR package. The pie chart of antibodies was created using the GGPLOT2 package and all the respective p-values in box plots are calculated using Wilcoxon test and stat_compare_means (paired = FALSE) function for respective condition pairs.

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The copyright holder for this preprint this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253367 doi: medRxiv preprint

We characterized the antibody titre, SARS-CoV-2 surrogate virus neutralization efficacy, cytokine levels, and single cell V(D)J profiles of 61 individuals (18 females and 43 males) distributed among three categories (Supplementary Figure 1A-B, Supplementary Table1 ). We performed ELISA from serum to quantify individual antibodies specific to spike RBD. The overall antibody distribution of contact showed increased levels of IgG and IgA in comparison to others but the median levels of IgM were similar to symptomatic ones, although higher than control (Supplementary Figure 1C) . For functional characterization, we determined the cytokine levels among the subject groups and found to be differentially secreted between contact and infected individuals. We observed that out of 48 cytokines 21 were able to distinguish between the two groups (data not shown) using logistic . 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 March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253367 doi: medRxiv preprint

The nature of protection observed in contact individuals is of great concern keeping in view the neutralizing efficacy towards SARS-CoV-2 antigen. The surged level of antibodies hints towards prior exposure with other pathogens having similar antigenic determinants. It is also exciting to note that when we correlated the antibody profile with the cytokine data, contacts showing elevated levels of antibodies were also showing high levels of particular cytokines. Eotaxin, G-CSF, IL-7 and MIP 1α are secreted across all subject groups in higher quantities than MIP 1-α which is a signature cytokine for increased disease severity. Among which, IL-7 and G-CSF play pivotal roles in lymphocyte expansion and rendering protection against invading pathogens respectively. The inflammatory chemokine Eotaxin and pro-inflammatory macrophage derived factor MIF are also key players in mediating immune responses. So, co-occurrence of specific cytokines with elevated Ig levels appeared to be a protective signature among contacts. Specifically, IgA antibody is of keen interest since it has the capacity of neutralizing respiratory viruses and protecting mucosal surfaces by hindering their attachment to epithelial cells [6] .

Direct contacts also showed specific BCR profiles and differential usage of genes along with unique clonotypes essential for high antibody production and cytokine secretion. All the above observations provided a strong and protective host-immune response as a major determining factor in contracting the COVID-19 disease.

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The copyright holder for this preprint this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253367 doi: medRxiv preprint

The blood samples for this study were obtained from TATA COVID hospital, Ganjam District, Odisha after due approval of the Institutional Biosafety and human ethics committee (Ref:

101/HEC/2020). For collecting the blood samples for the study due consent was taken from the subjects involved in the study.

This work is supported by the Department of Biotechnology, Government of India and the Odisha state government. . 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 March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253367 doi: medRxiv preprint 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)

The copyright holder for this preprint this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.11.21253367 doi: medRxiv preprint

Does the hygiene hypothesis apply to COVID-19 susceptibility? Microbes Infect

Prevalence of SARS-CoV-2 IgG antibodies in an area of northeastern Italy with a high incidence of COVID-19 cases: a population-based study

Prevalence of SARS-CoV-2 Antibodies in Health Care Personnel in the New York City Area

Distinct features of SARS-CoV-2-specific IgA response in COVID-19 patients

Mucosal Immunity in COVID-19: A Neglected but Critical Aspect of SARS-CoV-2 Infection

IgA dominates the early neutralizing antibody response to SARS-CoV-2

Serum IgA, IgM, and IgG responses in COVID-19

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The authors declare no conflict of interest.

We would like to thank all the volunteers who participated in the study voluntarily and provided their blood samples. We would also like to thank ILS core facilities (Single cell and genomics, Flow cytometry) and their staff for help and support. Also like to acknowledge ILS for institutional support for this study.. 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)