Within-host evolution of SARS-CoV-2 in an immunosuppressed COVID-19 patient as a source of immune escape variants

The origin of SARS-CoV-2 variants of concern remains unclear. Here, we test whether intra-host virus evolution during persistent infections could be a contributing factor by characterizing the long-term SARS-CoV-2 infection dynamics in an immunosuppressed kidney transplant recipient. Applying RT-qPCR and next-generation sequencing (NGS) of sequential respiratory specimens, we identify several mutations in the viral genome late in infection. We demonstrate that a late viral isolate exhibiting genome mutations similar to those found in variants of concern first identified in UK, South Africa, and Brazil, can escape neutralization by COVID-19 antisera. Moreover, infection of susceptible mice with this patient’s escape variant elicits protective immunity against re-infection with either the parental virus and the escape variant, as well as high neutralization titers against the alpha and beta SARS-CoV-2 variants, B.1.1.7 and B.1.351, demonstrating a considerable immune control against such variants of concern. Upon lowering immunosuppressive treatment, the patient generated spike-specific neutralizing antibodies and resolved the infection. Our results suggest that immunocompromised patients could be a source for the emergence of potentially harmful SARS-CoV-2 variants.


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All necessary data and informations are given in the manuscript. SARS-CoV-2 consensus sequences have been deposited to GISAID database (www.gisaid.org). Accession numbers are given in the manuscript (Supplementary table 2). Raw sequencing data have been submitted to the European Nucleotide Archive (https:// www.ebi.ac.uk/ena/browser) under the study accession number: ERP132087. EM structure of the closed conformation of D614G SARS-CoV-2 spike protein was loaded from the protein data bank (10.2210/pdb7BNM/pdb) and visualized with UCSF ChimeraX version: 1.1 (2020-09-09). In silico peptide binding was analyzed with ANN 4.0 on the Immune Epitope Database website (https://www.iedb.org/). For requests, please contact G. Kochs, georg.kochs@uniklinik-freiburg.de, and M. Panning, marcus.panning@uniklinik-freiburg.de. Requests will be processed within a week. Restrictions: Further additional information about the patient will not be shared due to protection of individuals' privacy.
-Sample sizes were not predictable in advance because of the limited number of immunosuppressed COVID-19 patients and convalescent COVID-19 patients that agreed to provide sera for this study.
-A single 58-years old male, kidney transplant patient was studied from March, 2020, to September 2020, at the University Hospital Freiburg.
-Immunocompetent patients were recruited and patient material was banked at the University Hospital Freiburg; inclusion criteria were: COVID-19 convalescent individuals following a mild to severe course of SARS-CoV-2 infection, SARS-CoV-2 infection was confirmed by positive PCR testing from oropharyngeal swab and/or SARS-CoV-2 spike IgG positive antibody testing.
-To show the immune escape of the patient's late isolate (d105) we used sera from 3 to 11 individuals for the different serological assays. The samples sizes were determined by the limited accessibility of sera from convalescent or vaccinated individuals early in the pandemic.
-For infection experiments with laboratory mice, 7-8 animals were used per group as recommended by the animal protection proposal approved by the local animal welfare committee. In the subsequent challenging experiments the numbers of animals were limited due to the small number of animals that survived the initial infection.
No data were excluded.
Data were reproduced as biological triplicates with technical duplicates in independent experiments.
-Accessible human antisera were choosen for the different serological assays according to the severity of COVID-19 or the history of vaccination to achive a diverse set of antisera. To analyze A*02/S133, A*02/S142, A*02/S244, A*03/S142 and A*03/S378-specific T cell responses participants needed to be allocated into experimental groups based on their HLA types (A*02 and A*03, respectively). In the present study, the covariates age and gender are not relevant for the experiments, as they have no direct influence on the experimental design to detect the presence of epitope-specific T cells.
-mice were allocated randomly concerning age and sex into the experimental groups.
Only objective parameters were included in the study design. Blinding was not applied. Non-objective parameters were not included in the study design. Due to standardized analyses of the flow cytometric data set, biased analysis can be excluded. all cell lines were tested monthly negative for mycoplasma no commonly misidentified cell lines were used in the study Transgenic (K18-hACE2)2Prlmn mice (Winkler et al., Nat Immunol. 2020 Nov;21(11):1327-1335. doi: 10.1038/s41590-020-0778-2. Epub 2020 Aug 24) were purchased from The Jackson Laboratory and bred locally. Hemizygous 8-12-week-old animals of both sexes were used. Mice were housed at 14-hour light/10-hour dark cycles and temperatures of~18-23°C with 40-60% humidity.

nature research | reporting summary
no wild animals were used in the study no field collected samples were used in the study All animal studies were performed in accordance with the guidelines of the Federation for Laboratory Animal Science Associations and the National Animal Welfare Body. All experiments were in compliance with the German animal protection law and approved by the animal welfare committee of the Regierungspraesidium Freiburg (permit G-20/91). The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Instrument Software Cell population abundance A single, 58-year-old male patient with a history of autosomal dominant polycystic kidney disease (ADPKD) was hospitalized at the University Medical Center, Freiburg, Germany. Population characteristics of the blood donors analyzed in figure 5 and supplementary figure 3: Two patients with severe COVID-19/ARDS, 50 and 61 years, male, used in panel 5a as positive control and in panel 5b, respectively; three patients with moderate COVID-19/O2 demand, 83-90 years, two males, one female, used in panel 5b, 5c, and 5d; twelve patients with mild COVID-19, 25-65 years, used in oanel 5b and suppl. fig. 3. Vaccine sera were from three healthy, female individuals, 31-46 years, used in panel 5b. Serum 11 in sup. fig.3 was from a male, in his fifties, after mild COVID-19 and a following Astra-Zeneca vaccination.
Diagnostic specimens of the immunosuppressed patient were analyzed in this study due to his long-term SARS-CoV-2 infection. Patients and vaccinees were recruited at the University Hospital Freiburg (in-and outpatient section); self-selection bias or other biases can be excluded since several people were included in the recruitment.
For T-cell analysis samples were banked and retrospectively selected according to the following inclusion criteria: HLA-A*02:01, HLA-A*03:01 The project has been approved by the University Medical Center, Freiburg, ethical committee. Written informed consent was obtained from all participants and the study was conducted according to federal guidelines, local ethics committee regulations (Albert-Ludwigs-Universität, Freiburg, Germany: No. F-2020-09-03-160428 and no. 322/20). All routine virological laboratory testing of patient specimens was performed in the Diagnostic Department of the Institute of Virology, University Medical Center, Freiburg (Local ethics committee no. 1001913).
Diagnostic specimens were collected between 20.03.20 to 28.09.20 during the standard care of the hospitalized patient. n.a.
Cryopreserved isolated human PBMCs were thawed and prepared for flow cytometry or in vitro expansion described in the methods section.