SARS-CoV-2 evolution during treatment of chronic infection

Abstract

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein¹ and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.

Extended Data Figure 1

Clinical time line of events with longitudinal respiratory sample CT values. CT – cycle threshold.

Extended data 2

A. Blood parameters over time in patient case: White cell count (WCC) and lymphocyte counts are expressed as x10³ Cells/mm. CRP: C reactive protein. B. Assessment of T cell and innate function. Whole blood cytokines were measured in whole blood after 24 hours stimulation either after T-cell stimulation with PHA or anti CD3/IL2 or innate stimulation with LPS. Healthy controls are shown as grey circles (N=15), Patient at d71 and d98 is shown as blue circles or red circles respectively. Cytokine levels are shown as pg/ml stimulation. Mean is shown by line and whiskers representing standard deviation.

Extended Data Figure 3

A. Serum SARS-CoV-2 antibody levels and virus population changes in chronic SARS-CoV-2 infection. Anti SARS-CoV2 IgG antibodies in patient and pre/post convalescent plasma compared to RNA+ Covid19 patients and prepandemic healthy controls: Red, grey and gold: IgG antibodies to SARS-CoV2 nucleocapsid protein (N), trimeric S protein (S) and the receptor binding domain (RBD) were measured by multiplexed particle based flow cytometry (Luminex) in RNA+ COVID-19 patients (N=20, red dots), Pre-pandemic healthy controls (N=20, grey dots) and in the convalescent donor plasma (orange dots); Results are shown as mean fluorescent intensity (MFI) +/- SD. Patient sera over time in blue: Anti SARS-CoV2 IgG to N (blue squares), S (blue circles) and RBD (blue triangles). Timing of CP units is also shown. B. SARS-CoV-2 antibody titres in patient and in convalescent plasma. Measurement of SARS-CoV-2 specific IgG antibody titres in three units of convalescent plasma (CP) by Euroimmun assay.

Extended data 4. Comparison between short-read (Illumina) and long-read single molecule (Oxford Nanopore) sequencing methods for the six observed Spike mutations.

Concordance was generally good between the majority of timepoints, however due to large discrepancies in a number of timepoints, we suggest that due to the high base calling error rate, Nanopore is not yet suitable for calling minority variants. As such, all figures in the main paper were produced using Illumina data only. B. Single genome sequencing (SGS) data from respiratory samples at indicated days. Indicated are the number of single genomes obtained at each time point with the mutations of interest (identified by deep sequencing). *denominator is 19 as for 2 samples the primer reads were poor quality at amino acid 796 at day 98. Amino acid variant and corresponding nucleotide position: S:W64G = 21752, S:Δ69 = 21765-21770, S:Y200H = 22160, S:T240I = 22281, S:P330S = 22550, S:D795H = 23948

Extended Data Figure 5. Evidence for within-host cladal structure.

A. Pairwise distances between samples measured using the all-locus distance metric plotted against pairwise distances in time (measured in days) between samples being collected. Internal distances between samples in the proposed main clade are shown in black, distances between samples in the main clade and samples collected on days 93 and 95 are shown in red, and internal distances between samples collected on days 93 and 95 are shown in green. B. Pairwise distances between samples in the larger clade (black) and between these samples and those collected on days 93 and 95 (red). The median values of the distributions of these values are significantly different according to a Mann Whitney test. C. Pairwise distances between samples in the main clade, once those collected on days 86, 89, 93, 95 have been removed (black) and between these samples and those collected on days 86 and 89 (red). The median values of the distributions of these values are not significantly different at the 5 level according to a Mann Whitney test.

Extended Data Figure 6

A. Close-view maximum-likelihood phylogenetic tree indicating the diversity of the case patient and three other long-term shedders from the local area (red, blue and purple), compared to recently published sequences from Choi et al (orange) and Avanzato et al (gold). Control patients generally showed limited diversity temporally, though the Choi et al sequences were highly divergent. Environmental samples (patient’s call bell, and patient’s mobile phone) are indicated. Tree branched have been collapsed where bootstrap support was <60. B. Highlighter plot indicating nucleotide changes at consensus level in sequential respiratory samples compared to the consensus sequence at first diagnosis of COVID-19. Each row indicates the timepoint the sample was collected (number of days from first positive SARS-CoV-2 RT-PCR). Black dashed lines indicate the RNA-dependent RNA polymerase (RdRp) and Spike regions of the genome. There were few nucleotide substitutions between days 1-54, despite the patient receiving two courses of remdesivir. The first major changes in the spike genome occurred on day 82, following convalescent plasma given on days 63 and 65. The amino acid deletion in S1, ΔH69/V70 is indicated by the black lines. Sites: Endotracheal aspirate (ETA) or Nose/throat swabs (N+T).

Extended Data 7. In vitro infectivity and neutralisation sensitivity of Spike pseudotyped lentiviruses.

A. infection of target 293T cells expressing TMPRSS2 and ACE2 receptors using equal amounts of virus as determined by reverse transcriptase activity. Data points represent technical replicates (n=2), with mean shown with error bars representing standard deviation. Data are representative of n=2 independent experiments (n=2). B. Representative Inverse dilution plots for Spike variants against convalescent plasma units 1-3. Data points represent mean neutralisation of technical replicates and error bars represent standard error of the mean of replicates. Data are representative of two independent experiments (n=2).

Extended Data Figure 8

A. Neutralization potency of a panel of monoclonal antibodies targeting the RBD is not impacted by Spike mutations D796H or ΔH69/V70. Lentivirus pseudotyped with SARS-CoV-2 Spike protein: WT (D614G background), D796H, ΔH69/V70, D796H+ΔH69/V70 were produced in 293T cells and used to infect target Hela cells stably expressing ACE2 in the presence of serial dilutions of indicated monoclonal antibodies. Data are means of technical replicates with error bars representing SD. Data are representative of at least two independent experiments. RBD: receptor binding domain. B. Classes of RBD binding antibodies and fold changes for Spike mutations D796H or ΔH69/V70 are indicated based Bouwer et al. Clusters II, V contain only non-neutralising mAbs, smaller neutralising mAb clusters IV (n=2) and X (n=1) were not tested. Red indicates significant fold changes.

Extended Data 9. Location of Spike mutations ΔH69/Y70 and D796H.

A. The SARS-CoV-2 spike trimer (PDB ID: 6xr8) with two protomers represented as surfaces and one protomer represented as a ribbon. The NTD is coloured in light blue, the RBD in light pink, the fusion peptide in dark pink, the HR1 domain in yellow, the CH domain in pale green, and the CD domain in brown. The location of D796 and H69 are indicated by red spheres. The loop connecting D796 to the fusion peptide is coloured magenta to improve visibility. The double grey lines provide orientation relative to the membrane. B. A close-up of the region defined by the box around H69 in panel A. H69 is highlighted in yellow. Residues containing atoms that are within 6 Å of H69 are highlighted in cyan. C. A close-up of the region defined by the box around D796 in panel A. D796 is highlighted in yellow. Residues containing atoms that are within 6 Å of D796 are highlighted in cyan. Hydrogen bonds are indicated by dashed yellow lines. Hydrophobic residues in the vicinity of D796 have been labelled. Y707 is from the neighbouring protomer. D. Global prevalence of selected spike mutations detailed in this paper. All high coverage sequences were downloaded from the GISAID database on 6^th January and aligned using MAFFT; as of this date there were 298254 sequences available. The global prevalence of each of the six spike mutations W64G, ΔH69/V70, Y200H, T240I, P330S and D796H were assessed by viewing the multiple sequence alignment in AliView, sorting by the column of interest, and counting the number of mutations.

Figure 1

Analysis of 23 Patient derived whole SARS-CoV-2 genome sequences in context of local sequences and other cases of chronic SARS-CoV-2 shedding. Circularised maximum-likelihood phylogenetic tree rooted on the Wuhan-Hu-1 reference sequence, showing a subset of 250 local SARS-CoV-2 genomes from GISAID. This diagram highlights significant diversity of the case patient (green) compared to three other local patients with prolonged shedding (blue, red and purple sequences). All “United Kingdom / English” SARS-CoV-2 genomes were downloaded from the GISAID database and a random subset of 250 selected as background.

Figure 2. Whole genome variant trajectories showing amino acids and relationship to treatments.

Data based on Illumina short read ultra deep sequencing at 1000x coverage. Variants shown reached a frequency of at least 10% in at least 2 samples. Treatments indicated are convalescent plasma (CP) and Remdesivir (RDV). Variants described in the text are designated by labels using the same colouring as the position in the genome. Variants labelled are represented by dashed lines. A. Variants detected in the patient from days 1-82. *D796H (light blue) is at the same frequency as NSP3 K902N (orange) therefore it is hidden beneath B. Variants detected in the patient from days 82-101.

Figure 3. Longitudinal variant frequencies and phylogenetic relationships for virus populations bearing six Spike (S) mutations

A. At baseline, all six S variants (Illumina sequencing) except for ΔH69/V70 were absent (<1% and <20 reads). Approximately two weeks after receiving two units of convalescent plasma (CP), viral populations carrying ΔH69/V70 and D796H mutants rose to frequencies >80% but decreased significantly four days later. This population was replaced by a population bearing Y200H and T240I, detected in two samples over a period of 6 days. These viral populations were then replaced by virus carrying W64G and P330S mutations in Spike, which both dominated at day 93. Following a 3^rd course of remdesivir and an additional unit of convalescent plasma, the ΔH69/V70 and D796H virus population re-emerged to become the dominant viral strain reaching variant frequencies of >75%. Pairs of mutations arose and disappeared simultaneously indicating linkage on the same viral haplotype. CT values from respiratory samples are indicated on the right y-axis (black dashed line and triangles). Where there were duplicate readings on the same day, to remain consistent, N+T samples were plotted B. Maximum likelihood phylogenetic tree of the case patient with day of sampling indicated. Spike mutations defining each of the clades are shown ancestrally on the branches on which they arose. On dates where multiple samples were collect, these are indicated as endotracheal aspirate (ETA) and Nose + throat swabs (N+T).

Figure 4. Spike mutant D796H + ΔH69/V70 infectivity and sensitivity convalescent plasma (CP).

A. western blot of virus pellets after centrifugation of supernatants from cells transfected with lentiviral pseudotyping plasmids including Spike protein. Blots are representative of two independent transfections. B. Single round Infectivity of luciferase expressing lentivirus pseudotyped with SARS-CoV-2 Spike protein (WT versus mutant) on 293T cells co-transfected with ACE2 and TMPRSS2 plasmids. Infectivity is corrected for reverse transcriptase activity in virus supernatant as measured by real time PCR. Data points represent technical replicates (n=3) with mean and error bars representing standard error of mean; data are representative of two independent experiments C-E. convalescent plasma (CP units 1-3) neutralization potency against pseudovirus virus bearing Spike mutants D796H, ΔH69/V70 and D796H + ΔH69/V70 F, G patient serum neutralisation potency against pseudovirus virus bearing Spike mutants D796H, ΔH69/V70 and D796H + ΔH69/V70. Patient serum was taken at indicated Day (D). Indicated is serum dilution required to inhibit 50% of virus infection (ID50), expressed as fold change relative to WT. Data points represent means of technical replicates and each data point is an independent experiment (n=2-6). Mean of data points in C-G is shown by horizontal bars.