key: cord-1019779-68sftpb5
authors: Vetter, P.; Eberhardt, C.; Meyer, B.; Martinez, P.; Torriani, G.; Pigny, F.; Lemeille, S.; Cordey, S.; Laubscher, F.; Vu, D.-L.; Calame, A.; Schibler, M.; Jacquerioz, F.; Blanchard, G.; Siegrist, C.-A.; Kaiser, L.; Didierlaurent, A.; eckerle, i.
title: Daily viral kinetics and innate and adaptive immune responses assessment in COVID-19: a case series
date: 2020-07-06
journal: nan
DOI: 10.1101/2020.07.02.20143271
sha: 6a18959e705df021aaf7a90eac1fe2de026f5144
doc_id: 1019779
cord_uid: 68sftpb5

Background Viral shedding patterns and its correlation with the immune responses of mildly symptomatic COVID-19 patients are still poorly characterized. Methods: We enrolled the first five COVID-19 patients quarantined in our institution; none received immunomodulatory treatment. We monitored shedding of viral RNA and infectious virus by RT-PCR and cell culture from the upper respiratory tract, and characterized the kinetics of systemic innate and adaptive immune responses. Results Despite mild clinical disease, high viral loads and shedding of infectious virus were observed from the respiratory tract, with isolation of infectious virus and prolonged positivity by PCR up to day 7 and 19 post onset of symptoms, respectively. Robust innate responses characterized by an increase in activated CD14+CD16+ monocytes and cytokine responses were observed as early as 2 days after symptoms onset. Cellular and humoral SARS-CoV-2 specific adaptive responses were detectable in all patients. Conclusion Infectious virus shedding was limited to the first week of symptom onset in mild cases. A strong innate response, characterized by the mobilization of activated monocytes during the first days of infection, as well as SARS-CoV-2 specific antibodies were detectable, even in patients with mild disease.

As of 18th June 2020, more than 8 million cases of COVID-19 have been reported worldwide, with 68 450,000 deaths [2] . While the first reports mainly described patients presenting with severe pneumonia 69 [3] , the spectrum of disease is wide, with more than 80% of infected individuals displaying moderate, mild 70 or even no symptoms [4, 5] .

Studies to date have focused on hospitalized patients with severe or critical disease. In those patients, 72 peak viral load in the upper respiratory tract occurs during the second week post onset of symptoms 73 (POS) , while viral clearance is achieved after 10 days in more than 90% of patients with mild disease [6] .

Elevated cytokine levels, including IL-6 and IP-10, increased CRP and profound T-cell lymphopenia 75 signal disease worsening [7] [8] [9] [10] . This dysregulated inflammatory response is thought to result from an 76 initial impairment of interferon production and thus reduced early viral control. Several reports have 77 suggested that SARS-CoV-2 antibody titers are higher in patients with more severe disease [3, 11] . 78 Activated SARS-CoV-2-specific CD4 and CD8 T cells have been reported in a few studies [12] [13] [14] . was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which this version posted July 6, 2020. . https: //doi.org/10.1101 //doi.org/10. /2020 

Study population and assessment 94 The first five patients (P1-P5) with COVID-19 confirmed by real-time reverse transcriptase polymerase 95 chain reaction (rtRT-PCR) and quarantined at HUG were included in this single-center prospective cohort.

These patients were screened for SARS-CoV-2 according to the national guidelines in place at the time, 97 which recommended screening travelers returning from Italy and reporting respiratory and/or flulike 98 symptoms. Ethical approval was waived by the local ethics committee (BASEC Req-2020-00143); written 99 informed consent for sample collection and coded data gathering was obtained from each patient. Clinical Concentrations of 24 markers (see supplement) were measured in cryopreserved plasma using a Luminex 121 assay (Magnetic Luminex assay, R&D Systems) according to the supplier's instructions. For data points 122 below the detection limit, a value of 50% of the last standard dilution value was assumed.

For phenotyping of blood cells, cryopreserved PBMCs were thawed, counted and divided to perform T-cell 124 and monocyte phenotyping. T cells were stained in PBS with LIVE/DEAD™ Stain Kit to exclude dead cells, 125 anti-CD3, -CD4, -CD8, -CD38 and anti-HLA-DR antibodies and were then fixed and permeabilized. Anti-126 Granzyme B and anti-Ki67 antibodies were used for intracellular staining. For phenotyping of monocytes, 127 cells were stained with FcR binding inhibitor, anti-CD3, -HLA-DR, -CD40, -CD123, -CD169, -CD20, -CCR2, 128 -CD14, -CD16, -CD86, -CD163 and anti-CCR7 antibodies. All data were acquired the same day on a 129 Fortessa II cytometer (BD Biosciences) and analyzed using FlowJo software (V10, Tree Star).

Antibody assays 132 S1 domain-specific IgG and IgA antibody response was measured using a commercially available kit 133 (Euroimmun AG, Lübeck, Germany, #EI 2606-9601 G and EI 2606-9601 A) according to the manufacturer's 134 instructions. To detect antibody against the complete S protein, ELISA plates were coated with a purified 135 trimerized S protein kindly provided by the Ecole polytechnique fédérale de Lausanne (EPFL). IgG, IgA and 136 IgM antibody titers were determined using a SARS-CoV-2 complete spike (S) protein-based rIFA [21, 22] . was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which this version posted July 6, 2020. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which this version posted July 6, 2020. . https://doi.org/10.1101/2020.07.02.20143271 doi: medRxiv preprint Full genome sequences from P1-4 were found to cluster with sequences representative of the closest 174 geographically related outbreak at that time, which was in Northern Italy. Molecular epidemiology is 175 consistent with histories of travel to the region shortly before symptom onset for P1, P2 and P3 ( Figure S3 ). was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which this version posted July 6, 2020. . https://doi.org/10.1101/2020.07.02.20143271 doi: medRxiv preprint 202 Antibody and cellular responses 203 Serum Ab titers against the spike protein (complete spike and S1 domain) were evaluated by ELISA and 204 rIFA assay (Figure 3 ). Patients 1, 3, 4 and 5 seroconverted by two weeks POS, including P1 who had very 205 mild and transient symptoms. For P2, no sample was available beyond day 7. P3 had no detectable IgA or 206 IgM, and only low IgG levels ( Figure 3A and B). Interestingly, this patient seroconverted only 3 weeks POS 207 in S1-based ELISA ( Figure 3A ), but had already seroconverted after two weeks in complete S-based assays 208 (rIFA and ELISA) ( Figure 3A and B) and in neutralization assays ( Figure was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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We report the kinetics of viral load and immune responses in the first five COVID-19 patients hospitalized 230 in our institution presenting with mild (P1-4) and severe (P5) disease. The most striking finding was a robust 231 innate response in patients with mild symptoms characterized by early activation of monocytes. We also 232 found prolonged viral RNA detection and measurable SARS-CoV-2 specific cellular and Ab responses 233 despite the paucity of symptoms. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which this version posted July 6, 2020. . https://doi.org/10.1101/2020.07.02.20143271 doi: medRxiv preprint is reported by Zhou and colleagues in ICU patients. The new observation could be due to the fact that our 258 study captured very early responses. The rapid mobilization of monocytes is indicative of a robust local 259 antiviral response. In patients without complications, this early mobilization of monocytes is not sustained 260 and rapidly declines to baseline one-week POS. This monocyte signature is not unique to SARS-CoV-2: it 261 has been described for dengue fever [35, 36] and other viral diseases [10, 37, 38] . was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which this version posted July 6, 2020. . https://doi.org/10.1101/2020.07.02.20143271 doi: medRxiv preprint interferon-dependent circulating cytokine responses. All patients mounted SARS-CoV-2 specific adaptive 286 responses including neutralizing antibodies. 287 All rights reserved. No reuse allowed without permission.

was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint (which this version posted July 6, 2020. . https://doi.org/10.1101/2020.07.02.20143271 doi: medRxiv preprint (A) IgG and IgA antibody kinetics for P1-5 using ELISA assays with either SARS-CoV-2 full S (only IgG, left 318 graph) or S1-domain (IgG and IgA, middle and right graph) as antigen. For Full S ELISA endpoint titers 319 were determined (starting dilution 1:100) whereas for S1-based ELISA the ratio of sample/calibrator was 320 measured (dilution 1:100). was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint (which this version posted July 6, 2020. . https: //doi.org/10.1101 //doi.org/10. /2020 with TRIzol (Invitrogen,Carlsbad, CA, USA) and resuspended in 10 μl of RNAse-free water. Ribosomal 552 RNA was removed using the Ribo-Zero Gold depletion kit (Illumina, San Diego, US). Thereafter, libraries 553 were generated using the TruSeq total RNA preparation protocol (Illumina) with dual indexing and loaded 554 on the HiSeq 4000 platform (Illumina) using the 2x100-bp protocol. Raw data were analysed as follows: 555 duplicate reads were removed using cd-hit (v4.6.8). Then reads were then trimmed to remove low-quality 556 and adapter sequences using Trimmomatic (v0.33). Next reads were mapped against the reference 557 sequences MN908947.3 using the SNAP nucleotide aligner program. was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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We coated 37.5ng/well of a purified trimerized S protein diluted in 0.1M sodium carbonate buffer pH 9.6 578 O/N at 4°C. Plates were blocked (5% milk PBS-T) and 100 ul of 2-fold serially diluted sera (range 100-579 12,800) were applied to wells with and without antigen. After incubation (1h at 37°C) and washing (3x 1min 580 with PBS-T) 100ul HRP-conjugated anti-human IgG (Jackson Immunoresearch, #109-036-098) diluted 581 1:16,000 was added. After incubation plates were washed and 100ul of TMB substrate (Invitrogen) was 582 added for 10min. Reaction was stopped by 100ul of 1N HCl and plates were read at 450nm. OD450 values 583 of blank wells were subtracted from values of antigen containing wells for each serum and each dilution.

The cut-off to determine the ELISA titer was set at 0.29 OD450, just before OD values reach the plateau was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint (which this version posted July 6, 2020. . https://doi.org/10.1101/2020.07.02.20143271 doi: medRxiv preprint 635 636 637 638 All rights reserved. No reuse allowed without permission.

was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint (which this version posted July 6, 2020. . https://doi.org/10.1101/2020.07.02.20143271 doi: medRxiv preprint 643 644 645 646 All rights reserved. No reuse allowed without permission.

was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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A pneumonia outbreak associated with a new coronavirus of probable 425 bat origin

Clinical features of patients infected with

The Lancet

Clinical features of covid-19

Characteristics of and Important Lessons From the

COVID-19) Outbreak in China: Summary of a Report of 433 72314 Cases From the Chinese Center for Disease Control and Prevention

Viral dynamics in mild and severe cases of COVID-19

Clinical and immunological features of severe and moderate 438 coronavirus disease 2019

Prediction models for diagnosis and prognosis of covid-19: 443 systematic review and critical appraisal

Hyper-activated pro-inflammatory CD16 monocytes correlate with the 445 severity of liver injury and fibrosis in patients with chronic hepatitis B. PLoS One

Impaired type I interferon activity and exacerbated inflammatory 448 responses in severe Covid-19 patients

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans 450 with COVID-19 Disease and Unexposed Individuals

Phenotype of SARS-CoV-2-specific T-cells in COVID-19 patients 455 with acute respiratory distress syndrome. medRxiv

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-462 PCR. Eurosurveillance

Comprehensive human virus screening using high-throughput 469 sequencing with a user-friendly representation of bioinformatics analysis: a pilot study

Validation of a commercially available SARS-CoV-2 serological 472 Immunoassay. medRxiv

per well and added on top of serum dilutions (final serum dilutions obtained were 606 from 1:10 to 1:2560). The virus-serum mix was incubated at 37°C, for 2h. Vero E6 were then infected with 607 100µl of virus-serum mixtures. After incubation at 37°C for 1.5h, cells were washed once with 1X PBS

After 16-20h of incubation at 37°C, 5% CO2 cells were fixed with 4% 609 formaldehyde solution for 15min at 37°C and nuclei stained with 1µg/ml DAPI solution. GFP positive 610 infected cells were counted with ImageXpress® Micro Widefield High Content Screening System (Molecular 611 Devices) and data analyzed

Epidemiology of viral 684 respiratory infections in a tertiary care centre in the era of molecular diagnosis

Faster and More 687 Accurate Sequence Alignment with SNAP

A vesicular stomatitis virus replicon-based bioassay for 689 the rapid and sensitive determination of multi-species type I interferon

Clotrimazole Derivatives as Specific Inhibitors of Arenavirus Fusion

Vesicular 695 stomatitis virus pseudotyped with severe acute respiratory syndrome coronavirus spike protein. 696 The Journal of general virology