2020/12/05 · cases of chronic SARS-CoV-2 shedding.. A

Neutralising antibodies drive Spike mediated SARS-CoV-2 evasion 1

2

Kemp SA1*, Collier DA1,2,3*, Datir R1,2,3, Gayed S4, Jahun A5, Hosmillo M5, Ferreira IATM2,3, Rees-3

Spear C1, Mlcochova P2,3, Ines Ushiro Lumb6, David Roberts6, Anita Chandra2,3, Temperton N7 , 4

The CITIID-NIHR BioResource COVID-19 Collaboration8, The COVID-19 Genomics UK (COG-UK) 5

Consortium9, Sharrocks K4, Blane E3, Briggs JAG10, van Gils MJ11, Smith KGC 2,3, Bradley JR3,12, 6

Smith C14, Goldstein RA1, Goodfellow IG5, Smielewska A5,13, Skittrall JP 4,14,15, Gouliouris T4, 7

Gkrania-Klotsas E4, Illingworth CJR14,16, McCoy LE1, Gupta RK2,3,16,17 8

9 1Division of Infection and Immunity, University College London, London, UK. 10 2 Cambridge Institute of Therapeutic Immunology & Infectious Disease (CITIID), Cambridge, UK. 11 3Department of Medicine, University of Cambridge, Cambridge, UK. 12 4Department of Infectious Diseases, Cambridge University NHS Hospitals Foundation Trust, 13

Cambridge, UK. 14 5Department of Pathology, University of Cambridge, Cambridge 15 6NHS Blood and Transplant, London, UK 16 7Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent, UK 17 8The CITIID-NIHR BioResource COVID-19 Collaboration, see appendix for author list 18 9The COVID-19 Genomics UK (COG-UK) Consortium, https://www.cogconsortium.uk. Full list of 19

consortium names and affiliations are in the appendix 20 10Medical Research Council Laboratory of Molecular Biology, Cambridge, UK. 21 11Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, 22

Amsterdam Institute for Infection and Immunity, Amsterdam, Netherlands 23 12 NIHR Cambridge Clinical Research Facility, Cambridge, UK. 24 13Department of Virology, Cambridge University NHS Hospitals Foundation Trust 25 14Department of Applied Mathematics and Theoretical Physics, University of Cambridge, UK 26 15Clinical Microbiology and Public Health Laboratory, Addenbrookes’ Hospital, Cambridge, UK 27 16 MRC Biostatistics Unit, University of Cambridge, Cambridge, UK 28 17Africa Health Research Institute, Durban, South Africa 29

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*equal contribution 30

31

Address for correspondence: 32

Ravindra K. Gupta 33

Cambridge Institute for Therapeutic Immunology and Infectious Diseases 34

Jeffrey Cheah Biomedical Centre 35

Puddicombe Way 36

Cambridge CB2 0AW, UK 37

Tel: +44 1223 331491 38

[email protected] 39

40

Key words: SARS-CoV-2; COVID-19; antibody escape, Convalescent plasma; neutralising 41

antibodies; mutation; evasion; resistance; immune suppression 42

43

Abstract 44

SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2, and amino acid 45

variation in Spike is increasingly appreciated. Given both vaccines and therapeutics are 46

designed around Wuhan-1 Spike, this raises the theoretical possibility of virus escape, 47

particularly in immunocompromised individuals where prolonged viral replication occurs. Here 48

we report fatal SARS-CoV-2 escape from neutralising antibodies in an immune suppressed 49

individual treated with convalescent plasma, generating whole genome ultradeep sequences by 50

both short and long read technologies over 23 time points spanning 101 days. Little 51

evolutionary change was observed in the viral population over the first 65 days despite two 52

courses of remdesivir. However, following convalescent plasma we observed dynamic virus 53

population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and 54

�H69/�V70 in the S1 NTD of the Spike protein. As serum neutralisation waned, viruses with the 55

escape genotype diminished in frequency, before returning during a final, unsuccessful course 56

of convalescent plasma. In vitro, the Spike escape variant conferred decreased sensitivity to 57

multiple units of convalescent plasma/sera from different recovered patients, whilst 58



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maintaining infectivity similar to wild type. These data reveal strong positive selection on SARS-59

CoV-2 during convalescent plasma therapy and identify the combination of Spike mutations 60

D796H and �H69/�V70 as a broad antibody resistance mechanism against commonly occurring 61

antibody responses to SARS-CoV-2. 62

63

Introduction 64

SARS-CoV-2 is an RNA betacoronavirus, with closely related viruses identified in pangolins and 65

bats1,2. RNA viruses have inherently higher rates of mutation than DNA viruses such as 66

Herpesviridae3. The capacity for successful adaptation is exemplified by the Spike D614G 67

mutation, that arose in China and rapidly spread worldwide4, now accounting for more than 68

90% of infections. The mutation appears to increase infectivity and transmissibility in animal 69

models5. Although the SARS-CoV-2 Spike protein is critical for virus infection via engagement of 70

ACE2, substantial Spike amino acid variation is being observed in circulating viruses6. Logically, 71

mutations in the receptor binding domain (RBD) of Spike are of particular concern due to the 72

RBD being targeted by neutralising antibodies and therapeutic monoclonal antibodies. 73

74

Deletions in the N-terminal domain (NTD) of Spike S1 are also being increasingly recognised, 75

both within hosts7 and across individuals8. The evolutionary basis for the emergence of 76

deletions is unclear at present but could be related to escape from immunity or to enhanced 77

fitness/transmission. The most notable deletion in terms of frequency is �H69/�V70. This 78

double deletion has been detected in multiple unrelated lineages, including the recent ‘Cluster 79

5’ mink related strain in the North Jutland region of Denmark (https://files.ssi.dk/Mink-cluster-80

5-short-report_AFO2). There it was associated with the RBD mutation Y453F in almost 200 81

individuals. Another European cluster in GISAID includes �H69/�V70 along with the RBD 82

mutation N439K. 83

84

Although �H69/�V70 has been detected multiple times, within-host emergence remains 85

undocumented and the reasons for its selection are unknown. Here we document real time 86

SARS-CoV-2 emergence of �H69/�V70 in response to convalescent plasma therapy in an 87



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immunocompromised human host, demonstrating broad antibody escape in combination with 88

the S2 mutation D796H. 89

90

Results 91

Clinical case history of SARS-CoV-2 infection in setting of immune-compromised host 92

Please contact authors for clinical details. Some indices are given in Supplementary table 1 and 93

Supplementary Figure 1 and Supplementary Figure 2). Figure 1 summarises the timing of 94

treatment. 95

96

Negative SARS-CoV-2 specific antibodies and requirement for convalescent plasma (CP) 97

Given the history of B cell depletion therapy and hypogammaglobulinemia we measured serum 98

SARS-CoV-2 specific antibodies over the course of the admission. Total serum antibodies to 99

SARS-CoV-2 were tested at days 44 and 50 by S protein immunoassay (Siemens). Results were 100

negative. Three units (200mL each) of convalescent plasma (CP) from three independent 101

donors were obtained from the NHS Blood and Transfusion Service and administered on 102

compassionate use basis. These had been assayed for antibody titres using the validated 103

Euroimmun assay (Supplementary figure 3). Patient serum was subsequently positive for SARS-104

CoV-2 specific antibodies by S protein immunoassay (Siemens) in the hospital diagnostic 105

laboratory on days 68, 90 and 101. 106

107

Virus genomic comparative analysis of 23 sequential respiratory samples over 101 days 108

The majority of samples were respiratory samples from nose and throat or endotracheal 109

aspirates during the period of intubation. Ct values ranged from 16-34 and all 23 respiratory 110

samples were successfully sequenced by standard long read approach as per the ARTIC protocol 111

implemented by COG-UK; of these 20 additionally underwent short-read deep sequencing using 112

the Illumina platform. There was generally good agreement between the methods, though as 113

expected nanopore demonstrated greater error at low variant frequencies (<10%) 114

(Supplementary Figure 4). We detected no evidence of recombination, based on two 115

independent methods. 116



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117

Maximum likelihood analysis of patient-derived whole genome consensus sequences 118

demonstrated clustering with other local sequences from the same region (Figure 2A). The 119

infecting strain was assigned to lineage 20B bearing the D614G Spike variant. Environmental 120

sampling showed evidence of virus on surfaces such as telephone and call bell but not in air on 121

days 59, 92 and 101. Sequencing of these surface viruses showed clustering with those derived 122

from the respiratory tract (Figure 2B). All samples were consistent with having arisen from a 123

single viral population. In our phylogenetic analysis, we included sequential sequences from 124

three other local patients identified with persistent viral RNA shedding over a period of 4 weeks 125

or more (Supplementary Table 2). Viruses from these individuals showed very little divergence 126

in comparison to the case patient (Figure 2B) and none showed amino acid changes in Spike 127

over time. We additionally inferred a maximum likelihood phylogeny comparing sequences 128

from these three local individuals and two long term immunosuppressed SARS-CoV-2 ‘shedders’ 129

recently reported7,9, (Figure 2B). While the sequences from Avanzato et al showed a pattern of 130

evolution more similar to two of the three other local patients, the case patient showed 131

significant diversification with a mutation rate of 30 per year (Supplementary table 2). 132

133

Further investigation of the sequence data suggested the existence of an underlying structure 134

to the viral population in our patient, with samples collected at days 93 and 95 being rooted 135

within, but significantly divergent from the original population (Figure 2B and 3A). The 136

relationship of the divergent samples to those at earlier time points rules out the possibility of 137

superinfection. The increased divergence of sequences does not necessarily indicate selection; 138

a spatially compartmentalised subset of viruses, smaller in number than the main viral 139

population, would be expected to evolve more quickly than the main population due to the 140

increased effect of genetic drift 10,11. 141

142

Virus population structure changes following convalescent plasma and remdesivir 143

All samples tested positive by RT-PCR and there was no sustained change in Ct values 144

throughout the 101 days following the first two courses of remdesivir (days 41 and 54), or the 145



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first two units of convalescent plasma (days 63 and 65). According to nanopore data, no 146

polymorphisms occurred over the first 60 days at consensus level (Figure 3A). However, short 147

read deep sequence Illumina data revealed minority polymorphisms in the viral population 148

during this period (Figure 3B). For example, T39I in ORF7a reached a majority frequency of 77% 149

on day 44, arising de novo and increased in frequency during the first period of the infection 150

(Supplementary Figure 6). 151

152

In contrast to the early period of infection, between days 66 and 82 a dramatic shift in virus 153

population structure was observed, with the near-fixation of D796H in S2 along with 154

�H69/�V70 in the S1 N-terminal domain (NTD) at day 82. This was identified in a nose and 155

throat swab sample with high viral load as indicated by Ct of 23 (Figure 4). The deletion was 156

not detected at any point prior to the day 82 sample, even as minority variants by short read 157

deep sequencing. 158

159

On Days 86 and 89, viruses collected were characterised by the Spike mutations Y200H and 160

T240I, with the deletion/mutation pair observed on day 82 having fallen to very low frequency. 161

Sequences collected on these days formed a distinct branch at the bottom of the phylogeny in 162

Figure 4 but were clearly associated with the remainder of the samples, suggesting that they 163

did not result from superinfection (Supplementary Figure 7), and further were not significantly 164

divergent from the bulk of the viral population (Supplementary Figure 5). 165

166

Sequencing of a nose and throat swab sample at day 93 again showed D796H along with 167

�H69/�V70 at <10% abundance, along with an increase in a virus population characterised by 168

Spike mutations P330S at the edge of the RBD and W64G in S1 NTD. This new lineage reached 169

near 100% abundance at day 93. Viruses with the P330S variant were detected in two 170

independent samples from different sampling sites, ruling out the possibility of contamination. 171

The divergence of these samples from the remainder of the population, noted above, suggests 172

the possibility of their resulting from the stochastic emergence, in the upper respiratory tract, 173

of a previously unobserved subpopulation of viruses (Supplementary Figure 5). 174



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175

Following the third course of remdesivir (day 93) and third CP (day 95), we observed a re-176

emergence of the D796H + �H69/�V70 viral population. The inferred linkage of D796H and 177

�H69/�V70 was maintained as evidenced by the highly similar frequencies of the two variants, 178

suggesting that the third unit of CP led to the re-emergence of this viral strain under renewed 179

positive selection. Ct values remained low throughout this period with hyperinflammation, 180

eventually leading to multi-organ failure and death at day 102. The repeated increase in 181

frequency of the novel viral strain during CP therapy strongly supports the hypothesis that the 182

deletion/mutation combination conferred antibody escape properties. 183

184

�H69/�V70 + D796H confers impaired neutralisation by multiple convalescent plasma units 185

and sera from recovered COVID-19 patients 186

Using lentiviral pseudotyping we expressed wild type and �H69/�V70 + D796H mutant Spike 187

protein in enveloped virions and compared neutralisation activity of CP against these viruses. 188

This system has been shown to give similar results to replication competent virus12,13. We first 189

tested infection capacity over a single round of infection and found that �H69/�V70 + D796H 190

had similar infectivity to wild type (both in a D614G background, Figure 5A, B). The �H69/�V70 191

+ D796H mutant was partially resistant to the first two CP units (Figure 5C, Table 1A). In 192

addition, patient derived serum from days 64 and 66 (one day either side of CP2 infusion) 193

similarly showed lower potency against the mutant (Figure 5C, Table 1A). The repeated 194

observation of D796H + �H69/�V70 emergence and positive selection strengthens the 195

hypothesis that these variants were the key drivers of antibody escape. Experimentally, the 196

D796H + �H69/�V70 mutant also demonstrated reduced susceptibility to the CP3 administered 197

on day 95, explaining its re-emergence (Figure 5C, Table 1A). 198

199

Given reduced susceptibility of the mutants to at least two units of CP, and the expansion of 200

sequences bearing �H69/�V70, we hypothesised that this represented a broad escape 201

mechanism. We therefore screened antiviral neutralisation activity in sera from five recovered 202

patients against the mutant and wild type viruses (Figure 5D). We observed that the mutant 203



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was indeed significantly less susceptible to four of five randomly selected sera, with the fifth 204

showing reduced susceptibility that did not reach statistical significance (Table 1B). Fold change 205

reductions in susceptibility of the mutant were as high as ten-fold compared to wild type (Table 206

1B). 207

208

In order to probe the impact of the D796H and �H69/�V70 mutations on potency of 209

monoclonal antibodies (mAbs) targeting Spike, we screened neutralisation activity of a panel of 210

seven neutralizing mAbs across a range of epitope clusters13 (Figure 5E). We observed no 211

differences in neutralisation between single mutants and wild type, suggesting that the 212

mechanism of escape was likely outside these epitopes. 213

214

In order to understand the mechanisms that might confer resistance to antibodies we 215

examined a published Spike structure and annotated it our residues of interest (Figure 6). This 216

analysis showed that �H69/�V70 is in a disordered, glycosylated loop at the very tip of the 217

NTD, and therefore could alter binding of antibodies. ΔH69/V70 is close to the binding site of 218

the polyclonal antibodies derived from COV57 plasma, indicating the sera tested here may 219

contain similar antibodies 14,15. D796H is in an exposed loop in S2 (Figure 6) and appears to be in 220

a region frequently targeted by antibodies16, despite mutations at position 796 being rare 221

(Supplementary table 4). 222

223

Discussion 224

Here we have documented a repeated evolutionary response by SARS-CoV-2 against antibody 225

therapy during the course of a persistent and eventually fatal infection in an 226

immunocompromised host. The observation of potential selection for specific variants 227

coinciding with the presence of antibodies from convalescent plasma is supported by the 228

experimental finding of reduced susceptibility of these viruses to plasma. Further, we were 229

able to document real-time emergence of a variant �H69/�V70 in the NTD of Spike that has 230

been increasing in frequency in Europe. In the case we report that it was not clear that the 231

emergence of the antibody escape variant was the primary reason for treatment failure. 232



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However, given that both vaccines and therapeutics are aimed at Spike, our study raises the 233

possibility of virus evasion, particularly in immune suppressed individuals where prolonged viral 234

replication occurs. 235

236

Our observations represent a very rare insight, and only possible due to lack of antibody 237

responses in the individual following administration of the B cell depleting agent rituximab for 238

lymphoma, and an intensive sampling course undertaken due to concerns about persistent 239

shedding and risk of nosocomial transmission. Persistent viral replication and the failure of 240

antiviral therapy allowed us to define the viral response to convalescent plasma, similar to a 241

recent report on asymptomatic long term shedding with four sequences over 105 days9, 242

although the reported shifts in genetic composition of the viral population could not be 243

explained phenotypically. Another common finding is the very low neutralisation activity in 244

serum post transfusion of CP with waning as expected. Apart from the difference in the 245

outcome of infection (severe, fatal disease versus asymptomatic disease and clearance), 246

critically important differences in our study include: 1. The intensity of sampling and use of both 247

long and short read sequencing to verify variant calls, thereby providing a unique scientific 248

resource for longitudinal population genetic analysis. 2. The close alignment between the 249

genetic composition of the viral population and CP administration, with an experimentally 250

verified resistant strain emerging, falling to low frequency, and then rising again under CP 251

selection. 3. Real time detection of emergence of a variant, �H69/�V70, that is increasing in 252

frequency in Europe, and shown here to affect neutralization by multiple COVID-19 patient 253

derived sera. 254

255

We have noted in our analysis the potential influence of compartmentalised viral replication 256

upon the sequences recovered in upper respiratory tract samples. Both population genetic and 257

small animal studies have shown a lack of reassortment between influenza viruses within a 258

single host during an infection, suggesting that acute respiratory viral infection may be 259

characterised by spatially distinct viral populations17,18. In the analysis of data, it is important to 260

distinguish genetic changes which occur in the primary viral population from apparent changes 261



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that arise from the stochastic observation of spatially distinct subpopulations in the host. While 262

the samples we observe on days 93 and 95 of infection are genetically distinct from the others, 263

the remaining samples are consistent with arising from a consistent viral population, supporting 264

the finding of a reversion and subsequent regain of antibody resistance. We note that in a 265

study of SARS-CoV-2, Choi et al reported the detection in postmortem tissue of viral RNA not 266

only in lung tissue, but also in the spleen, liver, and heart7. Mixing of virus from different 267

compartments, for example via blood, or movement of secretions from lower to upper 268

respiratory tract, could lead to fluctuations in viral populations at particular sampling sites. 269

Experiments in animal models with sampling of different replication sites could allow a better 270

understanding of SARS-CoV-2 population genetics and enable prediction of escape variants 271

following antibody based therapies. 272

273

This is a single case report and therefore limited conclusions can be drawn about 274

generalisability. 275

In addition to documenting the emergence of SARS-CoV-2 Spike �H69/�V70 + D796H in vivo, 276

conferring broad reduction in susceptibility to serum/plasma polyclonal, but no effect on a set 277

of predominantly RBD-targeting monoclonal antibodies, these data highlight that infection 278

control measures need to be specifically tailored to the needs of immunocompromised 279

patients. The data also highlight caution in interpretation of CDC guidelines that recommend 20 280

days as the upper limit of infection prevention precautions in immune compromised patients 281

who are afebrile19. Due to the difficulty with culturing clinical isolates, use of surrogates for 282

infectious virus such as sgRNA are warranted20. However, where detection of ongoing viral 283

evolution is possible, this serves as a clear proxy for the existence of infectious virus. In our case 284

we detected environmental contamination whilst in a single occupancy room and the patient 285

was moved to a negative-pressure high air-change infectious disease isolation room. 286

287

The clinical efficacy of CP has been called into question recently21, and our data suggest caution 288

in use of CP in patients with immune suppression of both T cell and B cell arms. In such cases, 289

the antibodies administered have little support from cytotoxic T cells, thereby reducing chances 290



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of clearance and raising the potential for escape mutations. Whilst we await further data, CP 291

administered for clinical need in immune suppression, should ideally be undertaken in single 292

occupancy rooms with enhanced infection control precautions, including SARS-CoV-2 293

environmental sampling and real-time sequencing. 294

295

296

Table 1: Neutralisation activity of convalescent plasma and patient sera taken in between CP 297

doses against SARS-CoV-2 viral variants observed in A. patient case. B. five unselected 298

patients with recovered COVID-19. Shown are IC50 dilution titres - the CP or serum reciprocal 299

dilution at which 50% of virus infectivity is lost against wild type (baseline) or D796H + Δ69/70 300

mutant Spike protein expressed on the surface of a lentiviral particle. Fold change is the 301

difference between the two viruses, and values below 1 indicate that the D796H/Δ69/70 302

mutant is less sensitive to the CP or serum. Only the D66 serum sample failed to demonstrate 303

significantly higher resistance of the D796H/Δ69/70 mutant. 304

305

A. IC50 as reciprocal dilution (95% CI)

WT D796H + Δ69/70 Fold Change IC50 (95% CI)

CP1 2480.0 (2104.0 - 2965.0) 924.2 (634.1 - 1316.0) 0.37 (0.24 - 0.56)

D64 serum 163.7 (106.6 - 256.4) 55.6 (29.5 - 100.6) 0.27 (0.12 - 0.77)

CP2 447.1 (305.2 - 670.1) 196 (129.8 - 294.7) 0.47 (0.25 - 0.86)

D66 serum 175.7 (90.7 - 410.4) 85.5 (48.2 - 149.8) 0.82 (0.29 - 1.96)

CP3 280.2 (172.8 - 480.3) 115 (75.0 - 175.5) 0.47 (0.24 - 0.91)

306

307

308

B. IC50 as reciprocal dilution (95% CI)

WT D796H + Δ69/70 Fold Change IC50 (95% CI)

Serum 1 994.1 (852.7-1159.0) 755.8 (426.7-1269.0) 0.71 (0.39-1.33)

Serum 2 2107.0 (1680.0-2662.0) 1045.0 (745.9-1387.0) 0.42 (0.27-0.70)



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Serum 3 3241.0 (2377.0-4643.0) 347.4 (234.4-505.4) 0.11 (0.07-0.17)

Serum 4 17368.0 (14357.0-22130.0) 2965.0 (2287.0-3767.0) 0.26 (0.20-0.32)

Serum 5 52735.0 (34276.0-93957.0) 3871.0 (3525.0-4252.0) 0.20 (0.17-0.24)

309

310

Ethics 311

The study was approved by the East of England – Cambridge Central Research Ethics Committee 312

(17/EE/0025). Written informed consent was obtained from both the patient and family. 313

Additional controls with COVID-19 were enrolled to the NIHR BioResource Centre Cambridge 314

under ethics review board (17/EE/0025). 315

316

Acknowledgements 317

We are immensely grateful to the patient and his family. We would also like to thank the staff 318

at CUH and the NIHR Cambridge Clinical Research Facility. We would like to thank Dr Ruthiran 319

Kugathasan and Professor Wendy Barclay for helpful discussions and Dr Martin Curran, Dr 320

William Hamilton, and Dr. Dominic Sparkes. We would like to thank Prof Andres Floto and Prof 321

Ferdia Gallagher. We thank Dr James Voss for the kind gift of HeLa cells stably expressing ACE2. 322

COG-UK is supported by funding from the Medical Research Council (MRC) part of UK Research 323

& Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research 324

Limited, operating as the Wellcome Sanger Institute. RKG is supported by a Wellcome Trust 325

Senior Fellowship in Clinical Science (WT108082AIA). LEM is supported by a Medical Research 326

Council Career Development Award (MR/R008698/1). SAK is supported by the Bill and Melinda 327

Gates Foundation via PANGEA grant: OPP1175094. DAC is supported by a Wellcome Trust 328

Clinical PhD Research Fellowship. CJRI acknowledges MRC funding (ref: MC_UU_00002/11). 329

This research was supported by the National Institute for Health Research (NIHR) Cambridge 330

Biomedical Research Centre, the Cambridge Clinical Trials Unit (CCTU) and by the UCL 331

Coronavirus Response Fund and made possible through generous donations from UCL’s 332

supporters, alumni, and friends (LEM). JAGB is supported by the Medical Research Council 333



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(MC_UP_1201/16). IG is a Wellcome Senior Fellow and supported by the Wellcome Trust 334

(207498/Z/17/Z). 335

336

Author contributions 337

Conceived study: RKG, SAK, DAC, AS, TG, EGK 338

Designed experiments: RKG, SAK, DAC, LEM, JAGB, EGK, AC, NT, AC, CS 339

Performed experiments: SAK, DAC, LEM, RD, CRS, AJ, IATMF, KS, TG, CJRI, BB, JS, MJvG 340

Interpreted data: RKG, SAK, DAC, PM, LEM, JAGB, PM, SG, KS, TG, JB, KGCS, IG, CJRI, JAGB, IUL, 341

DR, JS, BB, RAG 342

343

Conflicts of interest 344

The authors declare no conflicts of interest 345

346

Methods 347

Clinical Sample Collection and Next generation sequencing 348

Serial samples were collected from the patient periodically from the lower respiratory tract 349

(sputum or endotracheal aspirate), upper respiratory tract (throat and nasal swab), and from 350

stool. Nucleic acid extraction was done from 500µl of sample with a dilution of MS2 351

bacteriophage to act as an internal control, using the easyMAG platform (Biomerieux, Marcy-352

l'Étoile) according to the manufacturers’ instructions. All samples were tested for presence of 353

SARS-CoV-2 with a validated one-step RT q-PCR assay developed in conjunction with the Public 354

Health England Clinical Microbiology 22. Amplification reaction were all performed on a 355

Rotorgene™ PCR instrument. Samples which generated a CT of ≤36 were considered to be 356

positive. 357

358

Sera from recovered patients in the COVIDx study23 were used for testing of neutralisation 359

activity by SARS-CoV-2 mutants. 360

361



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For viral genomic sequencing, total RNA was extracted from samples as described. Samples 362

were sequenced using MinION flow cells version 9.4.1 (Oxford Nanopore Technologies) 363

following the ARTICnetwork V3 protocol (https://dx.doi.org/10.17504/protocols.io.bbmuik6w) 364

and BAM files assembled using the ARTICnetwork assembly pipeline 365

(https://artic.network/ncov-2019/ncov2019-bioinformatics-sop.html). A representative set of 366

10 sequences were selected and also sequenced using the Illumina MiSeq platform. Amplicons 367

were diluted to 2 ng/µl and 25 µl (50 ng) were used as input for each library preparation 368

reaction. The library preparation used KAPA Hyper Prep kit (Roche) according to manufacturer’s 369

instructions. Briefly, amplicons were end-repaired and had A-overhang added; these were then 370

ligated with 15mM of NEXTflex DNA Barcodes (Bio Scientific, Texas, USA). Post-ligation products 371

were cleaned using AMPure beads and eluted in 25 µl. Then, 20 µl were used for library 372

amplification by 5 cycles of PCR. For the negative controls, 1ng was used for ligation-based 373

library preparation. All libraries were assayed using TapeStation (Agilent Technologies, 374

California, USA) to assess fragment size and quantified by QPCR. All libraries were then pooled 375

in equimolar accordingly. Libraries were loaded at 15nM and spiked in 5% PhiX (Illumina, 376

California, USA) and sequenced on one MiSeq 500 cycle using a Miseq Nano v2 with 2x 250 377

paired-end sequencing. A minimum of ten reads were required for a variant call. 378

379

Bioinformatics Processes 380

For long-read sequencing, genomes were assembled with reference-based assembly and a 381

curated bioinformatics pipeline with 20x minimum coverage across the whole-genome 24. For 382

short-read sequencing, FASTQs were downloaded, poor-quality reads were identified and 383

removed, and both Illumina and PHiX adapters were removed using TrimGalore v0.6.6 25. 384

Trimmed paired-end reads were mapped to the National Center for Biotechnology Information 385

SARS-CoV-2 reference sequence MN908947.3 using MiniMap2-2.17 with arguments -ax and sr 386 26. BAM files were then sorted and indexed with samtools v1.11 and PCR optical duplicates 387

removed using Picard (http://broadinstitute.github.io/picard). Single nucleotide polymorphisms 388

(SNPs) were called using Freebayes v1.3.2 27 with a ploidy setting of 1, minimum allele 389

frequency of 0.20 and a minimum depth of five reads. Finally, a consensus sequences of nucleic 390



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acids with a minimum whole-genome coverage of at least 20× were generated with BCFtools 391

using a 0% majority threshold. 392

393

Phylogenetic Analysis 394

All available full-genome SARS-CoV-2 sequences were downloaded from the GISAID database 395

(http://gisaid.org/) 28 on 17th September. Duplicate and low-quality sequences (>5% N regions) 396

were removed, leaving a dataset of 138,472 sequences with a length of >29,000bp. All 397

sequences were sorted by name and only sequences sequenced with United Kingdom / England 398

identifiers were retained. From this dataset, a subset of 250 sequences were randomly 399

subsampled using seqtk (https://github.com/lh3/seqtk). These 250 sequences were aligned to 400

the 23 patient sequences, as well as three other control patients (persistent long-term shedders 401

from the same hospital) (Supplementary Table 2) and the SARS-CoV-2 reference strain 402

MN908947.3, using MAFFT v7.473 with automatic flavour selection 29. Major SARS-CoV-2 clade 403

memberships were assigned to all sequences using the Nextclade server v0.8 404

(https://clades.nextstrain.org/). 405

406

Maximum likelihood phylogenetic trees were produced using the above curated dataset using 407

IQ-TREE v2.1.2 30. Evolutionary model selection for trees were inferred using ModelFinder 31 408

and trees were estimated using the GTR+F+I model with 1000 ultrafast bootstrap replicates 32. 409

All trees were visualised with Figtree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/), rooted 410

on the SARS-CoV-2 reference sequence and nodes arranged in descending order. Nodes with 411

bootstraps values of <50 were collapsed using an in-house script. 412

413

Molecular substitution (clock) rates for the index patient, as well as three long-term shedders 414

and two recently described immunocompromised patients from literature, were estimated 415

using BEAST v2.6.3 34 using a HKY substitution model with 4 rate categories drawn from a 416

gamma distribution, a strict clock and a coalescent exponential population tree prior. MCMC 417

was run for 100 million iterations excluding a 15% burn-in. Tracer v1.7.1 was used to analyse 418

the BEAST trace in order extract the clock rate and ensure convergence had occurred. 419



https://doi.org/10.1101/2020.12.05.20241927


420

In-depth allele frequency variant calling 421

The SAMFIRE package35 was used to call allele frequency trajectories from BAM file data. Reads 422

were included in this analysis if they had a median PHRED score of at least 30, trimming the 423

ends of reads to achieve this if necessary. Nucleotides were then filtered to have a PHRED 424

score of at least 30; reads with fewer than 30 such reads were discarded. Distances between 425

sequences, accounting for low-frequency variant information, was also conducted using 426

SAMFIRE. The sequence distance metric, described in an earlier paper 11, combines allele 427

frequencies across the whole genome. Where L is the length of the genome, we define q(t) as a 428

4 x L element vector describing the frequencies of each of the nucleotides A, C, G, and T at each 429

locus in the viral genome sampled at time t. For any given locus i in the genome we calculate 430

the change in allele frequencies between the times t1 and t2 via a generalisation of the 431

Hamming distance 432

433

��, �� 12 |�� |��,,,��

434

where the vertical lines indicate the absolute value of the difference. These statistics were then 435

combined across the genome to generate the pairwise sequence distance metric 436

437

��, �� , ��

438

The Mathematica software package was to conduct a regression analysis of pairwise sequence 439

distances against time, leading to an estimate of a mean rate of within-host sequence 440

evolution. In contrast to the phylogenetic analysis, this approach assumed the samples 441

collected on days 93 and 95 to arise via stochastic emission from a spatially separated 442

subpopulation within the host, leading to a lower inferred rate of viral evolution for the bulk of 443

the viral population. 444

445



https://doi.org/10.1101/2020.12.05.20241927


Inference of selection 446

Under the assumption of a large effective population size, a deterministic one-locus model of 447

selection was fitted to genome sequence data describing changes in the frequency of the 448

variant T39I. Where q(t) represents the frequency of a single variant at time t, we used a 449

maximum likelihood method to infer the initial variant frequency at time t=1, and the selection 450

coefficient s, for times from the initial time point to the disappearance of the variant from the 451

population. Specifically, where n(ti) and N(ti) were the number of observations of the variant 452

and the total read depth at that locus at time ti, we fitted the model 453

454

��

�� 455

456

so as to maximise the binomial likelihood 457

458

� log �� 1 � ��

�

A similar calculation was performed to estimate the mean effective selection (incorporating 459

intrinsic selection for the variant plus linkage with other selected alleles) acting upon the 460

variant D796H during the final period of CP therapy. In this case selection was modelled as 461

being time-dependent, kicking in with the administration of therapy. We fitted the model: 462

463

��

1 � � �� ! �

464

to the data, setting τ = 95. 465

466

Recombination Detection 467

All sequences were tested for potential recombination, as this would impact on evolutionary 468

estimates. Potential recombination events were explored with nine algorithms (RDP, MaxChi, 469

SisScan, GeneConv, Bootscan, PhylPro, Chimera, LARD and 3SEQ), implemented in RDP5 with 470



https://doi.org/10.1101/2020.12.05.20241927


default settings 36. To corroborate any findings, ClonalFrameML v1.12 37 was also used to infer 471

recombination breakpoints. Neither programs indicated evidence of recombination in our data. 472

473

Structural Viewing 474

The Pymol Molecular Graphics System v2.4.0 (https://github.com/schrodinger/pymol-open-475

source/releases) was used to map the location of the four spike mutations of interested onto a 476

SARS-CoV-2 spike structure visualised by Wrobel et al (PDB: 6ZGE) 38. 477

478

Generation of Spike mutants 479

Amino acid substitutions were introduced into the D614G pCDNA_SARS-CoV-2_Spike plasmid 480

as previously described39 using the QuikChange Lightening Site-Directed Mutagenesis kit, 481

following the manufacturer’s instructions (Agilent Technologies, Inc., Santa Clara, CA). 482

483

Pseudotype virus preparation 484

Viral vectors were prepared by transfection of 293T cells by using Fugene HD transfection 485

reagent (Promega). 293T cells were transfected with a mixture of 11ul of Fugene HD, 1µg of 486

pCDNAΔ19Spike-HA, 1ug of p8.91 HIV-1 gag-pol expression vector40,41, and 1.5µg of pCSFLW 487

(expressing the firefly luciferase reporter gene with the HIV-1 packaging signal). Viral 488

supernatant was collected at 48 and 72h after transfection, filtered through 0.45um filter and 489

stored at -80˚C. The 50% tissue culture infectious dose (TCID50) of SARS-CoV-2 pseudovirus 490

was determined using Steady-Glo Luciferase assay system (Promega). 491

492

Serum/plasma pseudotype neutralization assay 493

Spike pseudotype assays have been shown to have similar characteristics as neutralisation 494

testing using fully infectious wild type SARS-CoV-212.Virus neutralisation assays were performed 495

on 293T cell transiently transfected with ACE2 and TMPRSS2 using SARS-CoV-2 Spike 496

pseudotyped virus expressing luciferase42. Pseudotyped virus was incubated with serial dilution 497

of heat inactivated human serum samples or convalescent plasma in duplicate for 1h at 37˚C. 498

Virus and cell only controls were also included. Then, freshly trypsinized 293T ACE2/TMPRSS2 499



https://doi.org/10.1101/2020.12.05.20241927


expressing cells were added to each well. Following 48h incubation in a 5% CO2 environment at 500

37°C, the luminescence was measured using Steady-Glo Luciferase assay system (Promega). 501

502

mAb pseudotype neutralisation assay 503

Virus neutralisation assays were performed on HeLa cells stably expressing ACE2 and using 504

SARS-CoV-2 Spike pseudotyped virus expressing luciferase as previously described43. 505

Pseudotyped virus was incubated with serial dilution of purified mAbs13 in duplicate for 1h at 506

37˚C. Then, freshly trypsinized HeLa ACE2- expressing cells were added to each well. Following 507

48h incubation in a 5% CO2 environment at 37°C, the luminescence was measured using Bright-508

Glo Luciferase assay system (Promega) and neutralization calculated relative to virus only 509

controls. IC50 values were calculated in GraphPad Prism. 510

511

Data Availability 512

Long-read sequencing data that support the findings of this study have been deposited in the 513

NCBI SRA database with the accession codes SAMN16976824 - SAMN16976846 under 514

BioProject PRJNA682013 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA682013). Short reads 515

and data used to construct figures were deposited at https://github.com/Steven-516

Kemp/sequence_files. 517

518

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622



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Figure 1

Figure 1. Analysis of 23 Patient derived whole SARS-CoV-2 genome sequences in context of national sequences and othercases of chronic SARS-CoV-2 shedding. A. Circularised maximum-likelihood phylogenetic tree rooted on the Wuhan-Hu-1reference sequence, showing a subset of 250 local SARS-CoV-2 genomes from GISAID. This diagram highlights significantdiversity of the case patient (green) compared to three other local patients with prolonged shedding (blue, red and purplesequences). All SARS-CoV-2 genomes were downloaded from the GISAID database and a random subset of 250 localsequences selected. B. Close-view maximum-likelihood phylogenetic tree indicating the diversity of the case patient andthree other long-term shedders from the local area (red, blue and purple), compared to recently published sequences fromChoi et al (orange) and Avanzato et al (gold). Control patients generally showed limited diversity temporally, though the Choiet al sequences were found to be even more divergent than the case patient. Environmental samples are indicated. 1000ultrafast bootstraps were performed and support at nodes is indicated.

2.0E-4

hCoV

-19/

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nd/C

AMB-

7FB5

6/20

20|E

PI_I

SL_4

3348

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NB15_CAMB-1B5CC9

hCoV-19/England/CAMB-1AABD9/2020|EPI_ISL_470190|2020-04-18|Europe

hCoV-19/England/CAMB-726F8/2020|EPI_ISL_439884|2020-03-24|Europe

hCoV-19/England/CAMB-77C79/2020|EPI_ISL_440815|2020-04-01|Europe

hCoV-19/England/CAMB-76BD7/2020|EPI_ISL_438431|2020-03-31|Europe

hCoV-19/England/CAMB-796C4/2020|EPI_ISL_441814|2020-04-03|EuropehC

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ngland/CAMB-843A1/2020|E

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urope

hCoV-19/England/CAMB-1AA2F2/2020|EPI_ISL_443682|2020-04-16|Europe

hCoV-19/England/CAMB-7D7BE/2020|EPI_ISL_442216|2020-04-07|Europe

hCoV-19/England/CAMB-1AA68D/2020|EPI_ISL_470215|2020-04-17|Europe

hCoV

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4289

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4-11

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hCoV-19/England/CAMB-72FFD/2020|EPI_ISL_440293|2020-03-21|Europe

hCoV-19/England/CAMB-8322D/2020|EPI_ISL_443485|2020-04-14|Europe

hCoV-19/England/CAMB-7DD16/2020|EPI_ISL_441669|2020-04-06|Europe

hCoV-19/England/CAMB-71B81/2020|EPI_ISL_439536|2020-03-31|Europe

hCoV-19/England/CAMB-73EA4/2020|EPI_ISL_440342|2020-03-20|Europe

hCoV-19/England/CAMB-1B194E/2020|EPI_ISL_452984|2020-05-13|Europe


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hCoV

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AMB-

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4-01

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hCoV-19/England/CAMB-7D94F/2020|EPI_ISL_441749|2020-04-05|Europe

hCoV-19/England/CAMB-7A0C5/2020|EPI_ISL_440612|2020-04-02|Europe

hCoV-19/England/CAMB-1AA553/2020|EPI_ISL_443604|2020-04-17|Europe

hCoV-19/England/CAMB-77ECE/2020|EPI_ISL_441840|2020-04-01|Europe

hCoV-19/England/CAMB-1AAB06/2020|EPI_ISL_470191|2020-04-18|Europe

hCoV-19/England/CAMB-1AA8C3/2020|EPI_ISL_470314|2020-04-17|Europe

hCoV-19/England/C

AMB-81544/2020|EPI_IS

L_442980|2020-04-12|Europe

hCoV-19/England/CAMB-7855F/2020|EPI_ISL_433747|2020-00-00|Europe

hCoV-19/England/CAMB-80ACE/2020|EPI_ISL_470388|2020-04-10|Europe

hCoV-19/England/CAMB-1AF1D2/2020|EPI_ISL_459273|2020-04-29|EuropehCoV-19/England/CAMB-7F28E/2020|EPI_ISL_442868|2020-04-08|Europe

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hCoV-19/England/CAMB-1B65DC/2020|EPI_ISL_584291|2020-10-02|Europe


NB14_CAMB-1B5CBA

hCoV-19/England/CAMB-843CF/2020|EPI_ISL_443445|2020-04-16|Europe

hCoV-19/England/CAMB-7EDBB/2020|EPI_ISL_442824|2020-04-06|Europe

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hCoV-19/England/CAMB-1ABFCD/2020|EPI_ISL_444356|2020-04-27|Europe

hCoV-19/England/CAMB-74CC7/2020|EPI_ISL_425364|2020-03-18|Europe

hCoV-19/England/CAMB-739A3/2020|EPI_ISL_425261|2020-00-00|Europe

hCoV-19/England/CAMB-1ACE74/2020|EPI_ISL_489505|2020-04-24|Europe

hCoV-19/England/C

AMB-1AF1B4/2020|EPI_ISL_459248|2020-04-29|Europe

hCoV-19/England/CAMB-73DB6/2020|EPI_ISL_433697|2020-04-01|Europe

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hCoV

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20|E

PI_I

SL_4

2534

5|202

0-03

-17|E

urop

e

hCoV-19/England/CAMB-1AFC3B/2020|EPI_ISL_459205|2020-05-03|Europe

hCoV-19/England/CAMB-82ADB/2020|EPI_ISL_438658|2020-04-23|Europe

hCoV

-19/

Engla

nd/C

AMB-

7EF2

E/20

20|E

PI_I

SL_4

4294

5|20

20-0

4-07

|Eur

ope

hCoV

-19/E

nglan

d/CAM

B-1A

E425

/2020

|EPI

_ISL_

4595

04|20

20-0

4-29

|Eur

ope


hCoV-19/England/CAMB-71C7F/2020|EPI_ISL_439650|2020-03-31|Europe


hCoV

-19/E

nglan

d/CAMB-83

876/2

020|E

PI_ISL_

4434

53|20

20-04

-15|E

urope


hCoV

-19/E

nglan

d/CAMB-7

50F7/2

020|E

PI_ISL_

4404

36|20

20-0

3-16

|Eur

ope

hCoV-19/England/CAMB-7C3CA/2020|EPI_ISL_434038|2020-04-14|Europe

hCoV-19/England/CAMB-727E6/2020|EPI_ISL_439580|2020-03-23|Europe

hCoV-19/England/CAMB-1AE355/2020|EPI_ISL_524592|2020-04-28|Europe

hCoV-19/England/CAMB-73FB0/2020|EPI_ISL_440355|2020-03-19|Europe

hCoV

-19/

Engla

nd/C

AMB-

82EA

2/20

20|E

PI_I

SL_4

4332

1|20

20-0

4-13

|Eur

ope

hCoV-19/England/CAMB-1B2CF9/2020|EPI_ISL_456720|2020-05-20


hCoV-19/England/C

AMB-7AC3A/2020|EPI_IS

L_433855|2020-04-08|Europe

NB01_CAMB-1B5124

hCoV-19/England/CAMB-8406E/2020|EPI_ISL_438729|2020-04-26|Europe


hCoV-19/England/CAMB-1B67B8/2020|EPI_ISL_584306|2020-10-03|Europe


hCoV-19/England/CAMB-7DED7/2020|EPI_ISL_470481|2020-04-06|Europe

hCoV-19/England/CAMB-7ED24/2020|EPI_ISL_442869|2020-04-07|Europe

hCoV-19/England/CAMB-1B1C27/2020|EPI_ISL_458562|2020-05-07|Europe


hCoV-19/England/CAMB-1ACF44/2020|EPI_ISL_489425|2020-04-24|Europe

hCoV-19/England/CAMB-7D129/2020|EPI_ISL_441655|2020-04-06|Europe

NB14_CAMB-1B5607NB07_CAMB-1B5A29

NB15_CAMB-1B43A7

hCoV-19/England/CAMB-76375/2020|EPI_ISL_438289|2020-03-28|EuropehCoV-19/England/CAMB-7A229/2020|EPI_ISL_441231|2020-04-02|Europe

hCoV

-19/

Engla

nd/C

AMB-

8239

4/20

20|E

PI_I

SL_4

4352

6|20

20-0

4-14

|Eur

ope

hCoV

-19/E

nglan

d/CAM

B-7B

8F9/2

020|E

PI_IS

L_44

2174

|2020

-04-

04|E

urop

e


hCoV-19/England/CAMB-744CF/2020|EPI_ISL_425298|2020-03-20|Europe

hCoV

-19/E

nglan

d/CAM

B-1A

B9CF

/2020

|EPI

_ISL_

4701

40|20

20-0

4-21

|Eur

ope

hCoV

-19/

Engla

nd/C

AMB-

8401

3/20

20|E

PI_I

SL_4

3872

4|20

20-0

4-26

|Eur

ope

NB02_CAMB-1B5AFChC

oV-1

9/Eng

land/C

AMB-

82A1

7/202

0|EPI

_ISL_

4386

47|20

20-0

4-23

|Eur

ope

hCoV-19/England/CAMB-1AED75/2020|EPI_ISL_452859|2020-05-07|Europe

hCoV

-19/

Engla

nd/C

AMB-

8407

D/20

20|E

PI_I

SL_4

3873

0|202

0-04

-26|E

urop

e

NB09_CAMB-1B0095

hCoV-19/England/CAMB-7830A/2020|EPI_ISL_433716|2020-00-00|Europe

hCoV-19/England/CAMB-7B3CB/2020|EPI_ISL_433950|2020-04-12|Europe


hCoV-19/England/CAMB-1AE07C/2020|EPI_ISL_459483|2020-04-27|Europe

MN908947.3

hCoV-19/England/CAMB-1AB6B9/2020|EPI_ISL_470324|2020-04-21|Europe

hCoV-19/England/CAMB-7377C/2020|EPI_ISL_425234|2020-03-29|Europe


hCoV

-19/

Engla

nd/C

AMB-

8158

0/20

20|E

PI_I

SL_4

4296

1|20

20-0

4-12

|Eur

ope

hCoV-19/England/CAMB-7EAA5/2020|EPI_ISL_442607|2020-04-06|Europe

hCoV-19/England/CAMB-1AE838/2020|EPI_ISL_524609|2020-04-30|EuropehC

oV-1

9/En

gland

/CAM

B-76

1D5/

2020

|EPI

_ISL

_438

452|

2020

-03-

30|E

urop

e

hCoV-19/England/CAMB-1B4A66/2020|EPI_ISL_473492|2020-06-07|Europe

hCoV-19/England/CAMB-79CA4/2020|EPI_ISL_440604|2020-04-02|Europe

hCoV-19/England/CAMB-7FE20/2020|EPI_ISL_444421|2020-04-19

hCoV-19/England/CAMB-77972/2020|EPI_ISL_439409|2020-03-31|EuropehCoV-19/England/CAMB-1B6897/2020|EPI_ISL_584310|2020-10-06|EuropehCoV-19/England/CAMB-7D2CC/2020|EPI_ISL_442267|2020-04-06|Europe

hCoV-19/England/CAMB-7C89E/2020|EPI_ISL_442102|2020-04-04|EuropeNB11_CAMB-1B5616



hCoV

-19/

Engla

nd/C

AMB-

7A33

5/20

20|E

PI_I

SL_4

4131

5|20

20-0

4-02

|Eur

ope


hCoV-19/England/CAMB-7CBC2/2020|EPI_ISL_442303|2020-04-04|Europe

hCoV-19/England/CAMB-823FE/2020|EPI_ISL_443449|2020-04-13|Europe

hCoV-19/England/CAMB-1AFD56/2020|EPI_ISL_452878|2020-05-11|Europe

hCoV-19/England/CAMB-1ACABC/2020|EPI_ISL_489479|2020-04-23|Europe

hCoV-19/England/CAMB-754DD/2020|EPI_ISL_425397|2020-03-17|EuropehCoV-19/England/CAMB-72142/2020|EPI_ISL_439660|2020-03-28|Europe

hCoV-19/England/CAMB-7EB39/2020|EPI_ISL_442917|2020-04-07|Europe

hCoV-19/England/CAMB-7D98B/2020|EPI_ISL_441683|2020-04-02|Europe

hCoV-19/England/CAMB-82CA8/2020|EPI_ISL_438678|2020-04-24|Europe



hCoV-19/England/CAMB-7D7CD/2020|EPI_ISL_441732|2020-04-06|Europe

NB02_CAMB-1B5BCC


hCoV-19/England/CAMB-7328A/2020|EPI_ISL_440267|2020-03-21|Europe


hCoV-19/England/CAMB-810BC/2020|EPI_ISL_438565|2020-04-19|Europe

hCoV-19/England/CAMB-82C3F/2020|EPI_ISL_438672|2020-04-24


hCoV-19/England/CAMB-1B0CBF/2020|EPI_ISL_452960|2020-05-12|Europe


hCoV-19/England/CAMB-7C05A/2020|EPI_ISL_433984|2020-00-00|Europe

hCoV-19/England/CAMB-7E98A/2020|EPI_ISL_442560|2020-04-07|Europe

NB06

_CAM

B-1A

DCF1

hCoV-19/England/CAMB-7F44C/2020|EPI_ISL_470359|2020-04-08|Europe

hCoV-19/England/CAMB-745DB/2020|EPI_ISL_425313|2020-03-28|Europe

hCoV-19/England/CAMB-1AE99F/2020|EPI_ISL_459286|2020-05-01|Europe



hCoV-19/England/C

AMB-1AF21B/2020|EPI_ISL_459322|2020-04-29|Europe



hCoV

-19/E

nglan

d/CAMB-79

71C/20

20|E

PI_ISL_

4410

88|20

20-04

-04|E

urope

hCoV

-19/

Engla

nd/C

AMB-

83E0

B/20

20|E

PI_I

SL_4

3869

6|20

20-0

4-25

|Eur

ope

hCoV-19/England/CAMB-7EB66/2020|EPI_ISL_442932|2020-04-07|Europe


hCoV-19/England/CAMB-83AE9/2020|EPI_ISL_443365|2020-04-15|Europe

NB20_CAMB-1B3654NB16_CAMB-1B19D5

hCoV-19/England/CAMB-1AD7F0/2020|EPI_ISL_448012|2020-05-04

hCoV

-19/

Engla

nd/C

AMB-

81DA

6/20

20|E

PI_I

SL_4

4259

0|20

20-0

4-13

|Eur

ope

hCoV

-19/

Engla

nd/C

AMB-

1AE5

31/2

020|

EPI_

ISL_

4594

60|2

020-

04-2

8|Eu

rope


hCoV-19/England/CAMB-7B9AB/2020|EPI_ISL_441612|2020-04-05|Europe

NB13_CAMB-1B5CE7


hCoV

-19/E

nglan

d/CAMB-8

3D95

/2020

|EPI_I

SL_43

8690

|2020

-04-

24|E

urop

e

hCoV-19/England/CAMB-7A8BE/2020|EPI_ISL_433826|2020-04-08|Europe

hCoV-19/England/CAMB-76C6B/2020|EPI_ISL_438257|2020-03-30|Europe

hCoV-19/England/CAMB-7228B/2020|EPI_ISL_439624|2020-03-28|Europe

hCoV-19/England/CAMB-77C3D/2020|EPI_ISL_440851|2020-03-31|Europe

hCoV

-19/

Engla

nd/C

AMB-

1B05

4B/2

020|

EPI_

ISL_

4528

90|2

020-

05-1

1|Eu

rope

hCoV-19/England/CAMB-7FD05/2020|EPI_ISL_444405|2020-04-19|Europe


hCoV-19/England/C

AMB-789F9/2020|EPI_ISL_433805|2020-04-07|Europe


hCoV-19/England/CAMB-1B01FC/2020|EPI_ISL_459304|2020-05-02|Europe

hCoV-19/England/CAMB-79AF5/2020|EPI_ISL_433823|2020-03-27|Europe

hCoV-19/England/CAMB-7CF5D/2020|EPI_ISL_442305|2020-04-06|Europe

hCoV-19/England/CAMB-73AFB/2020|EPI_ISL_425279|2020-03-30|Europe

hCoV-19/England/CAMB-1AC102/2020|EPI_ISL_444373|2020-04-29

hCoV-19/England/CAMB-1B2093/2020|EPI_ISL_453003|2020-05-18


hCoV-19/England/CAMB-7EF79/2020|EPI_ISL_442923|2020-04-07|Europe

hCoV-19/England/CAMB-7E61A/2020|EPI_ISL_524548|2020-04-08|Europe

NB08_CAMB-1B2CBD

hCoV-19/England/CAMB-1AE70E/2020|EPI_ISL_524583|2020-04-30|Europe hCoV

-19/E

nglan

d/CAMB-8

0CB9/2

020|E

PI_ISL_

4429

54|20

20-0

4-04

|Eur

ope

hCoV-19/England/CAMB-7F5B2/2020|EPI_ISL_442921|2020-04-08|Europe

hCoV-19/England/CAMB-83EDE/2020|EPI_ISL_438707|2020-04-25|Europe





hCoV-19/England/CAMB-1AC463/2020|EPI_ISL_489438|2020-04-22|Europe

hCoV

-19/

Engla

nd/C

AMB-

7C4F

4/20

20|E

PI_I

SL_4

4224

8|20

20-0

4-04

|Eur

ope

NB06_CAMB-1B55CE


hCoV-19/England/CAMB-80DF2/2020|EPI_ISL_442991|2020-04-10|Europe

hCoV-19/England/CAMB-7DF4D/2020|EPI_ISL_441602|2020-04-06|Europe


hCoV-19/England/CAMB-7A65A/2020|EPI_ISL_441303|2020-04-01|Europe



hCoV-19/England/CAMB-734DF/2020|EPI_ISL_440107|2020-03-21|Europe





NB09_CAMB-1B5C50

hCoV

-19/

Engla

nd/C

AMB-

726E

9/20

20|E

PI_I

SL_4

3987

9|202

0-03

-23|E

urop

e

NB01_CAMB-1B5ADE

hCoV-19/England/CAMB-7DA88/2020|EPI_ISL_441624|2020-04-06|Europe


hCoV-19/England/CAMB-1AA9A2/2020|EPI_ISL_470199|2020-04-18|Europe

hCoV

-19/

Engla

nd/C

AMB-

1ACF

CC/2

020|

EPI_

ISL_

4895

29|2

020-

04-2

4|Eu

rope




NB17_CAMB-1B5B71


hCoV-19/England/CAMB-1B2DE7/2020|EPI_ISL_477792|2020-05-18|Europe

NB10_CAMB-1B5643




NB10_CAMB-1B5C6F

hCoV-19/England/CAMB-1ACDE0/2020|EPI_ISL_489450|2020-04-23|Europe

hCoV-19/England/CAMB-75C8A/2020|EPI_ISL_438262|2020-03-29|Europe

hCoV-19/England/CAMB-7622D/2020|EPI_ISL_438366|2020-03-30|Europe

NB16_CAMB-1B5BAE hCoV-19/England/CAMB-1B102B/2020|EPI_ISL_459229|2020-05-05|Europe



hCoV-19/England/C

AMB-1AC427/2020|EPI_IS

L_489465|2020-04-21|Europe




NB08_CAMB-1B529A

hCoV-19/England/CAMB-1B5BCC/2020|EPI_ISL_529236|2020-08-15|Europe [&!color=#008026]

hCoV-19/England/CAMB-78A9C/2020|EPI_ISL_433811|2020-04-06|Europe

hCoV

-19/E

nglan

d/CAMB-7

3D7A

/2020

|EPI_I

SL_43

3693

|2020

-04-

01|E

urop

e


hCoV-19/England/CAMB-82AAE/2020|EPI_ISL_438655|2020-00-00|Europe

hCoV-19/England/CAMB-77EFB/2020|EPI_ISL_440826|2020-03-31|Europe

hCoV-19/England/CAMB-825DA/2020|EPI_ISL_443430|2020-04-12|Europe

hCoV

-19/

Engla

nd/C

AMB-

71D9

A/20

20|E

PI_I

SL_4

3830

0|20

20-0

3-30

|Eur

ope

hCoV

-19/

Engla

nd/C

AMB-

815D

B/20

20|E

PI_I

SL_4

4301

7|20

20-0

4-10

|Eur

ope

hCoV

-19/E

nglan

d/CAM

B-75

BAB/

2020

|EPI

_ISL_

4383

07|20

20-0

3-28

|Eur

ope

hCoV-19/England/CAMB-720EB/2020|EPI_ISL_439629|2020-03-28|Europe

hCoV

-19/

Engla

nd/C

AMB-

1AD2

B3/2

020|

EPI_

ISL_

4479

86|2

020-

00-0

0|Eu

rope

hCoV-19/England/CAMB-7EA4B/2020|EPI_ISL_442553|2020-04-07|Europe


NB02_CAMB-1B0AA6

hCoV

-19/

Engla

nd/C

AMB-

8320

F/20

20|E

PI_I

SL_4

4339

8|202

0-04

-14|E

urop

e

hCoV-19/England/CAMB-7DEF5/2020|EPI_ISL_470494|2020-04-07|Europe


hCoV

-19/

Engla

nd/C

AMB-

1AAC

5E/2

020|E

PI_I

SL_4

7018

7|202

0-04

-19|E

urop

e

hCoV-19/England/CAMB-7D60C/2020|EPI_ISL_442129|2020-04-07|Europe

hCoV-19/England/CAMB-1ADF61/2020|EPI_ISL_459475|2020-04-26|Europe

NB23_CAMB-1B4FB2

hCoV

-19/E

nglan

d/CAMB-7

84DA/20

20|E

PI_ISL_

4337

42|20

20-0

4-06

|Eur

ope


NB05_CAMB-1B55FB

hCoV-19/England/CAMB-1AA6D8/2020|EPI_ISL_470277|2020-04-17|Europe

NB16_CAMB-1B5C9C

NB01_CAMB-1B5BDB

hCoV-19/England/CAMB-1AD8EE/2020|EPI_ISL_448022|2020-05-02|Europe

hCoV

-19/E

nglan

d/CAMB-81

7E4/2

020|E

PI_ISL_

4431

12|20

20-04

-11|E

urope

hCoV-19/England/CAMB-7DDF8/2020|EPI_ISL_470381|2020-04-07|Europe

hCoV

-19/

Engla

nd/C

AMB-

83E1

A/20

20|E

PI_I

SL_4

3869

7|202

0-04

-26|E

urop

e

hCoV

-19/

Engla

nd/C

AMB-

7832

8/20

20|E

PI_I

SL_4

3371

8|20

20-0

4-04

|Eur

ope

hCoV-19/England/CAMB-7DC37/2020|EPI_ISL_441686|2020-04-06|Europe

hCoV-19/England/CAMB-1AEA23/2020|EPI_ISL_524584|2020-04-27|Europe

hCoV-19/England/CAMB-7AECB/2020|EPI_ISL_433889|2020-04-10|EuropehC

oV-1

9/En

glan

d/CA

MB-8

0897

/202

0|EP

I_IS

L_44

2895

|202

0-04

-09|

Euro

pe

NB08_CAMB-1B5467

hCoV

-19/E

nglan

d/CAM

B-81

1AA/

2020

|EPI

_ISL_

4385

77|20

20-0

4-20

|Eur

ope


hCoV

-19/E

nglan

d/CAMB-7

A159/2

020|E

PI_ISL_

4405

86|20

20-0

4-03

|Eur

ope

2.0E-4

A

B

2.0E-4

7 8

8 8

8 6

9 0

9 0

9 8

8 5

100

6 4

8 58 4

100

9 4

9 5

100

7 691

9 0

9 0

9 4

9 9

9 3

9 9

100

9 6

8 8

9 5

100

8 9

100

Wuhan-Hu-1

Patient’s Mobile Phone

Patient’s Call Bell

Case PatientControl Patient 1Control Patient 2Choi et al , NEJM (2020)Control Patient 3Avanzato et al , Cel l (2020)



https://doi.org/10.1101/2020.12.05.20241927


ETAETA

ETAETA

ETA

Figure 2. Virus genetics and population structure in chronic SARS-CoV-2 infection. A. Highlighter plot indicating nucleotidechanges 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). Blackdashed lines indicate the RNA-dependent RNA polymerase (RdRp) and Spike regions of the genome. Of particular interest, atconsensus level, there were no nucleotide substitutions on days 1-57, 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. Thedouble amino acid deletion in S1, ΔH69/V70 is indicated by black lines. All samples are nose and throat swabs unless indicatedwith ETA (endotracheal aspirate). B. Whole genome amino acid variant trajectories based on Illumina short read ultra deepsequencing at 1000x coverage. All variants which reached a frequency of at least 10% in at least two samples were plotted.Black dashed line represents Δ69/70. CP, convalescent plasma; RDV, Remdesivir.

1374145505455565766828689939395989999100100101101

ORF3a

ORF1ab Spike N

Envelope

ORFs 6-8

Membrane

ORF10

RdRp

ORF1ab Spike N

1000

0

1500

0

2000

0

2500

0

3000

0

50001

A C T G

Nucleotide Position

RdRp

Days

from

firs

t pos

itive

SAR

S-Co

V-2

RT-P

CRdel

A

B

Figure 2

CP1,2 CP3 + RDVRDV RDV

1 21 41 61 81 101Days since 1st Positive RT-PCR for SARS-CoV-2



https://doi.org/10.1101/2020.12.05.20241927


Figure 3

Figure 3. Longitudinal variant frequencies and phylogenetic relationships for virus populations bearing six Spike (S)mutations A. At baseline, all four S variants (Illumina sequencing) were absent (<1% and <20 reads). Approximately twoweeks after receiving two units of convalescent plasma (CP), viral populations carrying ΔH69/V70 and D796H mutants rose tofrequencies >90% but decreased significantly four days later. This population was replaced by a population bearing Y200Hand T240I, detected in two samples over a period of six days. These viral populations were then replaced by virus carryingW64G and P330S mutations in Spike, which both reached near fixation at day 93. Following a 3rd course of remdesivir and anadditional unit of convalescent plasma, the ΔH69/V70 and D796H virus population re-emerged to become the dominant viralstrain reaching variant frequencies of >90%. Pairs of mutations arose and disappeared simultaneously indicating linkage onthe same viral haplotype. B.Maximum likelihood phylogenetic tree of the case patient with day of sampling indicated. Spikemutations defining each of the clades are shown ancestrally on the branches on which they arose. Number at node denotessupport by ultrafast bootstrapping consisting of 1000 replicates.

A

B

6.0E-5

7 6

100

7 8

9 3

75

9 8

61

6 6

6 4

6 9

81

8 8

Wuhan-Hu-1

Day 37

Day 56

Day 45

Day 54

Day 55

Day 57

Day 1

Day 45

Day 95Day 93

Day 93

Day 50

Day 100

Day 82

Day 99

Day 101

Day 101

Day 100

Day 98

Day 99

Day 66

Day 89

Day 86

W64G, P330S

D614G

Y200H, T240IW258S

H69/V70, D796HΔ

1 371s

t Rem

desi

vir 4

1 45 502n

d Re

mde

sivi

r 54

1st +

2nd

CP

65 66 82 86 89

3rd

Rem

desi

vir 9

3

3rd

CP 9

5 98 99 100

1010

10

20

30

40

50

60

70

80

90

100

Number of days since 1st positive SARS-COV-2 Swab RT-PCR

Mut

atio

n Pr

evel

ance

(%)

D796HΔH69/V70 Y200H T240IP330SW64G

15

20

25

30

Ct



https://doi.org/10.1101/2020.12.05.20241927


Figure 4: Spike mutant D796H + ΔH69/V70 has infectivity comparable to wild type but is less sensitive tomultiple units of convalescent plasma (CP) and sera from recovered individuals. A. Reverse transcriptase activityof virus supernatants containing lentivirus pseudotyped with SARS-CoV-2 Spike protein (WT versus mutant) B.Single round Infectivity of luciferase expressing lentivirus pseudotyped with SARS-CoV-2 Spike protein (WT versusmutant) on 293T cells co-transfected with ACE2 and TMPRSS2 plasmids. C. Patient serum (taken at indicated Day(D) ) and convalescent plasma (CP units 1-3) neutralization potency against Spike mutant D796H + ΔH69/V70measured using lentivirus pseudotyped with SARS-CoV-2 Spike protein (WT versus mutant). Indicated is serumdilution required to inhibit 50% of virus infection. D. Neutralization potency of sera from 5 unselectedconvalescent patients (with previous confirmed SARS-CoV-2 infection) against WT versus mutant virus as in panelC. E Neutralization potency of a panel of monoclonal antibodies against lentivirus pseudotyped with SARS-CoV-2Spike protein (WT Wuhan versus single mutants D614G, D796H, ΔH69/V70). Data are representative of at leasttwo independent experiments * p<0.05

WT

WT+D79

6H+∆

69∆7

00

20,000

40,000

60,000

80,000

100,000

Infe

ctiv

ity

(RLU

/µL)

WT

WT+D79

6H+∆

69∆7

00

500,000

1,000,000

1,500,000

2,000,000

RT A

ctiv

ity

(pU

/µL)

A B

C D

E

Figure 4

CP1 D64 CP2 D66 CP30

1000

2000

3000

Sera

IC50

(Neu

tral

izat

ion)

WT

D796H/Δ69/70

S 5 S 4 S 3 S 2 S 10

100020003000

2000030000400005000060000

IC50

(Neu

tral

izat

ion)

WT

D796H/Δ69/70

CP1 D64 CP2 D66 CP30

1000

2000

3000

Sera

IC50

(Neu

tral

izat

ion)

WT

D796H/Δ69/70

* **

* * *

**

WT WTΔ69/70D796H

Δ69/70D796H

-4 -3 -2 -1 0-25

0

25

50

75

100

Log10 IgG µg/ml

% N

eutr

aliz

atio

n

COVA1-18

-3 -2 -1 0 1-25

0

25

50

75

100

Log10 IgG µg/ml

% N

eutr

aliz

atio

n

COVA1-12

-3 -2 -1 0 1-25

0

25

50

75

100

Log10 IgG µg/ml

% N

eutr

aliz

atio

n

COVA2-29

-3 -2 -1 0 1 2-25

0

25

50

75

100

Log10 IgG µg/ml

% N

eutr

aliz

atio

n

COVA2-02

-3 -2 -1 0 1-25

0

25

50

75

100

Log10 IgG µg/ml

% N

eutr

aliz

atio

n

COVA1-16

-3 -2 -1 0 1-25

0

25

50

75

100

Log10 IgG µg/ml

% N

eutr

aliz

atio

n

Δ6970

COVA1-21

D796HWT (D614G)

WT (Wuhan)

-3 -2 -1 0 1-25

0

25

50

75

100

Log10 IgG µg/ml

% N

eutr

aliz

atio

n

COVA2-07



https://doi.org/10.1101/2020.12.05.20241927


Figure 5. Location of Spike mutations ΔH69/Y70 in S1 and D796H in S2. Amino acid residues H69 andY70 deleted in the N-terminal domain (red) and D796H in subunit 2 (orange) are highlighted on a SARS-CoV-2 spike trimer (PDB: 6ZGE Wrobek et al., 2020). Each of the three protomers making up the Spikehomotrimer are coloured separately in shades of grey (centre). Close-ups of ΔH69/Y70 (above) andD796H (below) are shown in cartoon, stick representation. Both mutations are in exposed loops.

S1

S2

RBD

Viral Membrane

NTD

ΔH69/V70

D796

Figure 5

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