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. 2021 Feb 22;10(2):e1241. doi: 10.1002/cti2.1241

Human neutralising antibodies elicited by SARS‐CoV‐2 non‐D614G variants offer cross‐protection against the SARS‐CoV‐2 D614G variant

Cheryl Yi‐Pin Lee 1,2, Siti Naqiah Amrun 1,2, Rhonda Sin‐Ling Chee 1,2, Yun Shan Goh 1,2, Tze‐Minn Mak 3,4, Sophie Octavia 3,4, Nicholas Kim‐Wah Yeo 1,2, Zi Wei Chang 1,2, Matthew Zirui Tay 1,2, Anthony Torres‐Ruesta 1,2,5, Guillaume Carissimo 1,2, Chek Meng Poh 1,2, Siew‐Wai Fong 1,2,6, Wang Bei 2, Sandy Lee 2, Barnaby Edward Young 3,7,8, Seow‐Yen Tan 9, Yee‐Sin Leo 3,7,8,10, David C Lye 3,7,8,10, Raymond TP Lin 4,11, Sebastien Maurer‐Stroh 1,3,4,6,12, Bernett Lee 2, Cheng‐I Wang 2, Laurent Renia 1,2, Lisa FP Ng 1,2,5,13,14,✉
PMCID: PMC7899292  PMID: 33628442

Abstract

Objectives

The emergence of a SARS‐CoV‐2 variant with a point mutation in the spike (S) protein, D614G, has taken precedence over the original Wuhan isolate by May 2020. With an increased infection and transmission rate, it is imperative to determine whether antibodies induced against the D614 isolate may cross‐neutralise against the G614 variant.

Methods

Antibody profiling against the SARS‐CoV‐2 S protein of the D614 variant by flow cytometry and assessment of neutralising antibody titres using pseudotyped lentiviruses expressing the SARS‐CoV‐2 S protein of either the D614 or G614 variant tagged with a luciferase reporter were performed on plasma samples from COVID‐19 patients with known D614G status (n = 44 infected with D614, n = 6 infected with G614, n = 7 containing all other clades: O, S, L, V, G, GH or GR).

Results

Profiling of the anti‐SARS‐CoV‐2 humoral immunity reveals similar neutralisation profiles against both S protein variants, albeit waning neutralising antibody capacity at the later phase of infection. Of clinical importance, patients infected with either the D614 or G614 clade elicited a similar degree of neutralisation against both pseudoviruses, suggesting that the D614G mutation does not impact the neutralisation capacity of the elicited antibodies.

Conclusions

Cross‐reactivity occurs at the functional level of the humoral response on both the S protein variants, which suggests that existing serological assays will be able to detect both D614 and G614 clades of SARS‐CoV‐2. More importantly, there should be negligible impact towards the efficacy of antibody‐based therapies and vaccines that are currently being developed.

Keywords: clade, COVID‐19, cross‐reactivity, D614G variant, neutralising antibodies, SARS‐CoV‐2

A single point mutation from aspartic acid (D) to glycine (G) at position 614 of the SARS‐CoV‐2 spike (S) protein, termed D614G, has garnered global attention due to the observed increase in transmissibility and infection rate. Given that a majority of the developing antibody‐mediated therapies and serological assays are based on the S antigen of the original Wuhan reference sequence, it is crucial to determine whether humoral immunity acquired from the original SARS‐CoV‐2 isolate is able to induce cross‐detection and cross‐protection against the novel prevailing D614G variant. In this study, we demonstrated an overall equivalent neutralising capacity against both the D614 and G614 pseudoviruses, suggesting negligible impact towards the efficacy of antibody‐based therapies and vaccines that are currently being developed.

graphic file with name CTI2-10-e1241-g002.jpg

Introduction

Coronavirus disease 2019 (COVID‐19) is the consequence of an infection by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), which emerged in Wuhan, China, in December 2019. 1 The rapid expansion of the COVID‐19 pandemic has affected 213 countries and territories, with a global count of more than 80 million laboratory‐confirmed human infection cases to date. 2 An inevitable impact of this pandemic is the accumulation of immunologically relevant mutations among the viral populations due to natural selection or random genetic drift, resulting in enhanced viral fitness and immunological resistance. 3 , 4 For instance, antigenic drift was previously reported in other common cold coronaviruses, OC43 and 229E, as well as in SARS‐CoV. 5 , 6 , 7

In early March 2020, a non‐synonymous mutation from aspartic acid (D) to glycine (G) at position 614 of SARS‐CoV‐2 spike (S) protein was identified. 8 This variant, G614, rapidly became the dominant SARS‐CoV‐2 clade in Europe by May 2020, suggesting a higher transmission rate over the original isolate, D614. 8 In vitro and animal studies have also indicated that the G614 variant may have an increased infectivity and may be associated with higher viral loads and more severe infections. 8 , 9 , 10 , 11 , 12 Notably, single point mutations have been shown to induce resistance to neutralising antibodies in other coronaviruses, including SARS‐CoV and Middle East respiratory syndrome (MERS‐CoV). 13 , 14 More importantly, mutations in the S protein of SARS‐CoV‐2 have been shown to induce conformational modifications that alter antigenicity. 15 , 16 Hence, determining any cross‐neutralising capability of antibodies developed against the earlier G614 variant is of paramount importance to validate the therapeutic efficacy of developing immune‐based interventions.

Results

Antibody profiling against the SARS‐CoV‐2 S protein was first assessed using plasma samples collected from COVID‐19 patients (n = 57) during the Singapore outbreak between January and April 2020, across the early recovery phase [median 31 days post‐illness onset (pio)] and a later post‐recovery time point (median 98 days pio) (Table 1, Figure 1a and b). All patients showed a decrease in IgM response (Figure 1a), and a prolonged IgG response over time (Figure 1b). Although one recent study has demonstrated similar neutralisation profiles against both D614 and G614 SARS‐CoV‐2 pseudoviruses, the virus clade by which the six individuals were infected with was not identified. 9 According to Singapore’s SARS‐CoV‐2 clade pattern from December 2019 till July 2020 based on n = 736 cases with genome availability, the D614G mutation, indicated as G clade following the GISAID clade nomenclature, only appeared in March 2020 (Figure 1c). Hence, with knowledge on the D614G status of a subset of COVID‐19 patients (n = 44 infected with D614, n = 6 infected with G614, n = 7 containing all other clades: O, S, L, V, G, GH or GR; Table 1, Figure 1c), the neutralising capacity of these anti‐SARS‐CoV‐2 antibodies was assessed using pseudotyped lentiviruses expressing the SARS‐CoV‐2 S protein tagged with a luciferase reporter as a surrogate of live virus. 17 The neutralisation EC50 values of each patient were interpolated from the respective dose–response neutralisation titration curves (Table 2, Figure 1d and e, Supplementary figure 1). Notably, these antibodies were able to neutralise both SARS‐CoV‐2 D614 and G614 pseudoviruses at similar levels, despite having a significantly lower neutralisation capacity at median 98 days pio in all COVID‐19 patients (Figure 1d and e, Supplementary figures 1 and 2). Corroborating other studies, severe patients have a higher and persisting level of neutralising antibodies as compared with both mild and moderate patients (Table 2, Supplementary figure 2). 18 , 19 Of clinical importance, all the patients infected with either the D614 or G614 clade elicited a similar degree of neutralisation against both D614 and G614 pseudoviruses (Figure 1f), suggesting that the D614G mutation does not impact the neutralisation capacity of the elicited antibodies. Our results support the notion that the locus where the point mutation occurred is not critical for antibody‐mediated immunity and may not have an impact on virus resistance towards antibody‐based interventions. 4 , 20

Table 1.

Demographic and clinical information of COVID‐19 patients

Patients (n = 57)
Demographics
Age, years 45 (13)
Sex
Male 38 (66.7%)
Female 19 (33.3%)
Ethnicity
Chinese 42 (73.7%)
Others 15 (26.3%)
Comorbidities 29 (50.9%)
Hyperlipidaemia 14 (24.6%)
Hypertension 13 (22.8%)
Diabetes 7 (12.3%)
Myocardial infection (history) 5 (8.8%)
Others 10 (17.5%)
D614G infection status
D614 44 (77.2%)
G614 6 (10.5%)
Others a 7 (12.3%)
Clinical outcome (clinical severity; group)
No pneumonia (0; mild) 25 (43.9%)
Pneumonia, without hypoxia (1; moderate) 19 (33.3%)
Pneumonia, with hypoxia (2; severe) 13 (22.8%)
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Data are presented as Mean (SD) or n (%). COVID‐19: Coronavirus Disease 2019.

a

Others: O, S, L, V, G, GH or GR clades.

Figure 1.

Figure 1

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Timeline of events during the SARS‐CoV‐2 outbreak in Singapore, and the antibody profiles of COVID‐19 patients and their neutralising capacity against both D614 and G614 variants of SARS‐CoV‐2. Plasma samples of COVID‐19 patients (n = 57) at median 31 and median 98 days post‐illness onset (pio) were assessed for anti‐SARS‐CoV‐2 IgM and IgG antibody response. Plasma samples (1:100 dilution) were incubated with transduced HEK293T cells expressing SARS‐CoV‐2 spike protein, and (a) anti‐IgM and (b) anti‐IgG levels were quantified by flow cytometry. Percentage binding indicates the percentage of cells with antibody binding. Data are shown as mean ± SD of two independent experiments. Dotted line indicates mean + 3SD of healthy controls (n = 22). Statistical analysis was carried out with the Wilcoxon signed‐rank test (*P < 0.05, ***P < 0.001). (c) Percentage of COVID‐19 cases with genome available (n = 736) during the Singapore outbreak from December 2019 to July 2020, segregated by the clade with which the patients were infected following GISAID clade nomenclature. (d–f) Anti‐SARS‐CoV‐2 neutralising antibodies were assessed using luciferase expressing lentiviruses pseudotyped with SARS‐CoV‐2 spike (S) protein of either the original strain, D614, or the mutant variant, G614. Log10 neutralisation EC50 profiles against (d) D614 and (e) G614 pseudoviruses across both time points. Data represent the mean of two independent experiments, and statistical analysis was carried out using the paired t‐test (***P < 0.001). (f) Comparison of log10 neutralisation EC50 values between D614 and G614 pseudoviruses during both time points. Data represent the mean of two independent experiments, and statistical analysis was carried out using the paired t‐test. All data points are non‐significant (ns).

Table 2.

Neutralisation EC50 values of COVID‐19 patients

Patient Days post‐illness onset (pio) Recovery phase Infection by SARS‐CoV‐2 strain a D614 (EC50) Dilution factor D614 (Log 10 EC50) Dilution factor G614 (EC50) Dilution factor G614 (Log 10 EC50) Dilution factor
Mild (No pneumonia)
#1 39 Early Others 93.821 1.972300058 27.088 1.432776941
95 Late 36.481 1.562066734 ND ND
#2 34 Early D614 59.67 1.775756038 59.527 1.774713996
152 Late 59.156 1.7719988 46.489 1.667350204
#3 30 Early D614 84.26 1.925621455 100.33 2.001430812
111 Late 36.216 1.558900481 20.109 1.303390474
#4 29 Early D614 264.7 2.422753941 371.63 2.570110765
92 Late 85.178 1.930327439 101.03 2.004450353
#5 30 Early D614 401.03 2.603176862 229.98 2.36169007
100 Late 93.083 1.968870372 42.272 1.626052796
#6 32 Early D614 56.708 1.753644331 49.807 1.697290384
96 Late 37.541 1.574505837 24.87 1.395675785
#7 30 Early D614 182.16 2.260453018 179.26 2.253483392
107 Late 37.299 1.571697188 31.102 1.492788317
#8 30 Early D614 70.715 1.849511546 64.52 1.809694359
88 Late 38.049 1.580343247 32.853 1.516575034
#9 25 Early D614 61.803 1.791009557 67.785 1.8311336
101 Late 45.326 1.656347394 13.3 1.123851641
#10 32 Early D614 123.21 2.090645958 72.937 1.862947896
110 Late 18.353 1.263707065 ND ND
#11 33 Early D614 312.72 2.495155657 135.08 2.130591052
91 Late 103.42 2.014604533 60.652 1.782845126
#12 33 Early D614 365.85 2.563303059 233.92 2.369067355
96 Late 79.832 1.90217701 35.665 1.552242228
#13 31 Early G614 110.63 2.043872912 127.51 2.105544246
94 Late 65.001 1.812920038 63.342 1.801691772
#14 24 Early D614 151.32 2.179896333 143.27 2.156155261
100 Late 39.825 1.600155784 31.445 1.497551599
#15 28 Early D614 242.06 2.383923029 241.44 2.382809222
98 Late 58.31 1.765743041 52.821 1.722806619
#16 31 Early D614 169.39 2.228887768 134.4 2.128399269
92 Late 78.702 1.895985769 78.239 1.893423291
#17 39 Early D614 89.4 1.951337519 77.364 1.888538916
97 Late 25.104 1.399742926 14.494 1.161188257
#18 26 Early D614 16.219 1.210024074 13.513 1.130751777
99 Late ND ND ND ND
#19 39 Early G614 18.721 1.272329043 24.532 1.389732956
99 Late 10.11 1.004751156 17.581 1.245043574
#20 35 Early D614 941.37 2.973760354 856.37 2.932661445
99 Late 171 2.23299611 97.95 1.99100444
#21 35 Early D614 312.28 2.494544171 150.83 2.178487731
99 Late 38.602 1.586609806 19.899 1.298831252
#22 32 Early G614 17.385 1.240174695 18.098 1.257630584
98 Late 83.448 1.921415932 74.848 1.8741802
#23 62 Early G614 36.553 1.562923026 31.281 1.495280628
104 Late 24.869 1.395658322 29.766 1.473720477
#24 38 Early D614 10.477 1.020236944 ND ND
99 Late ND ND ND ND
#25 18 Early D614 849.23 2.929025328 ND ND
105 Late 601.69 2.779372794 ND ND
Moderate (Pneumonia, without hypoxia)
#1 29 Early D614 325.6 2.512684396 311.41 2.493332555
99 Late 50.013 1.699082906 40.54 1.607883744
#2 29 Early Others 280.08 2.447282098 279.51 2.44639735
91 Late 55.82 1.746789832 49.937 1.698422448
#3 37 Early D614 565.39 2.752348123 412.73 2.615666037
99 Late 176.37 2.246424715 192.41 2.28422764
#4 29 Early D614 406.93 2.609519708 394.6 2.596157081
92 Late 58.04 1.763727404 70.882 1.850535963
#5 29 Early D614 188.21 2.274642695 172.03 2.235604189
106 Late 197.85 2.296336055 157.28 2.1966735
#6 25 Early D614 2349.4 3.370956964 2000.3 3.301095135
96 Late 432.12 2.635604367 319.05 2.503858749
#7 34 Early D614 96.242 1.983364639 110.53 2.04348017
104 Late 10.932 1.038699623 12.366 1.092229242
#8 28 Early D614 227 2.356025857 215.24 2.332922983
113 Late 41.09 1.613736141 28.984 1.462158321
#9 31 Early D614 792.61 2.899059547 601.93 2.779545989
96 Late 182.48 2.261215272 132.86 2.123394248
#10 32 Early D614 541.77 2.733814953 399.85 2.6018971
99 Late 136.61 2.135482491 121.88 2.085932446
#11 29 Early D614 164.37 2.215822555 152.3 2.182699903
90 Late 34.63 1.539452492 41.678 1.61990687
#12 32 Early D614 241.37 2.38268329 267.15 2.426755179
89 Late 35.053 1.544725193 39.4 1.595496222
#13 58 Early D614 84.158 1.925095406 51.315 1.710244333
101 Late 34.56 1.538573734 25.507 1.406659382
#14 25 Early D614 220.86 2.344117068 171.07 2.233173855
106 Late 31.918 1.50403567 33.142 1.520378713
#15 36 Early D614 200.82 2.302806963 156.64 2.194902674
87 Late 70.748 1.849714167 65.35 1.815245592
#16 27 Early D614 308.07 2.488649409 201.4 2.304059466
106 Late 90.322 1.955793546 56.963 1.755592854
#17 34 Early D614 1079.6 3.033262876 1039.5 3.016824494
115 Late 100.36 2.001560653 119.98 2.079108858
#18 42 Early D614 89.823 1.953387556 69.059 1.839220285
107 Late 31.172 1.493764668 31.425 1.497275286
#19 30 Early G614 214.79 2.332014058 186.07 2.269676358
99 Late 54.362 1.735295426 38.613 1.586733545
Severe (Pneumonia, with hypoxia)
#1 31 Early G614 740.24 2.869372549 548.74 2.739366619
92 Late 154.05 2.187661703 92.754 1.967332648
#2 33 Early Others 940.91 2.973548084 967.53 2.98566444
97 Late 250.17 2.398235229 199.92 2.300856243
#3 29 Early D614 1597.5 3.203440867 1443.9 3.159537116
96 Late 173.92 2.240349527 236.97 2.374693369
#4 29 Early D614 970.61 2.987044761 651.53 2.813934418
104 Late 106.39 2.026900809 86.982 1.939429389
#5 34 Early D614 755.31 2.878125235 822.44 2.915104224
113 Late 71.959 1.857085119 74.804 1.873924822
#6 33 Early Others 2042.2 3.310098272 2007.9 3.30274208
110 Late 100.71 2.003072596 108.06 2.033664963
#7 30 Early D614 1291.7 3.11116166 3109.8 3.492732459
87 Late 420.78 2.624055089 996.85 2.998629813
#8 28 Early D614 1298.1 3.11330815 1391.8 3.143576832
109 Late 224.08 2.350403096 246.4 2.391640703
#9 37 Early Others 466.49 2.668842338 383.24 2.583470831
92 Late 156.93 2.195705975 140.67 2.148201487
#10 39 Early Others 4453.3 3.648681953 3528.8 3.547627045
116 Late 1024.2 3.010384771 1072.7 3.030478281
#11 40 Early D614 529.25 2.723660867 730.88 2.863846078
60 Late 253.5 2.403977964 419.99 2.62323895
#12 31 Early D614 891.98 2.950355117 1016.9 3.007278247
93 Late 136.02 2.133602771 108.15 2.034026524
#13 40 Early Others 1595.2 3.202815141 1691.3 3.228220649
60 Late 612.24 2.7869217 702.75 2.846800854
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COVID‐19: Coronavirus Disease 2019; Early: median 31 days post‐illness onset (pio); Late: median 98 days pio; ND: not determined.

a

Others: O, S, L, V, G, GH or GR clades.

Discussion

The emergence of a new virus clade due to random mutations could heavily deter the therapeutic outcome of treatments and vaccines. Majority of the current immunoassays developed against SARS‐CoV‐2 are based on the S antigen of the original Wuhan reference sequence. 21 , 22 Moreover, pioneer batches of therapeutics and candidate vaccines were mostly designed based on earlier infections. As a result, mutations in the dominant variant sequence could potentially alter the viral phenotype and virulence, thereby rendering current immune‐based therapies less efficient and effective. 23 , 24 Fortunately, a recent pre‐print reported no observable difference in IgM, IgG and IgA profiles against either the D614 or G614 S variant in an antigen‐based serological assay, 25 providing preliminary findings on the effectiveness of current diagnostic approaches to detect SARS‐CoV‐2 G614 infections.

In addition, determining the level of cross‐reactivity is essential for immunosurveillance, as well as to identify broadly neutralising antibodies or epitopes. 26 Here, we confirm that cross‐reactivity occurs at the functional level of the humoral response on both the S protein variants. Of note, the stronger neutralising capacity observed during the early recovery phase may be due to the higher level of IgM response at median 31 days pio, as plasma IgM has been shown in a recent pre‐print to contribute towards SARS‐CoV‐2 neutralisation. 27 While IgA has also been reported to mediate neutralising activities during SARS‐CoV‐2 infection at a lower potency, 27 investigations on the IgA levels and neutralising capacity in patients infected by the G614 clade would be needed to confirm earlier findings. Interestingly, although there was no significant difference between the neutralising capacity against both D614 and G614 pseudoviruses, individuals infected by the G614 clade, albeit small patient numbers, appear to have a lower log10 EC50 value (Figure 1d–f). While it remains elusive, this observation may be associated to the lower IgM and IgG levels in these patients. Nonetheless, our results, together with the recent serological evaluation, 25 strongly suggest that existing serological assays will be able to detect both D614 and G614 clades of SARS‐CoV‐2 with a similar sensitivity. Recent studies have also demonstrated an overall equivalent sensitivity against both the D614 and G614 pseudotyped viruses, suggesting that the D614G mutation is not expected to hinder current vaccine development. 10 , 11 , 12 , 28 However, it is of clinical relevance to assess if cross‐reactivity between the variants may enhance viral infection when neutralising antibodies are present at suboptimal concentrations. 29 More importantly, further studies using monoclonal antibodies are necessary to validate the cross‐reactivity profiles between both SARS‐CoV‐2 S variants.

Overall, our study shows that the D614G mutation on the S protein does not impact SARS‐CoV‐2 neutralisation by the host antibody response, nor confer viral resistance against the humoral immunity. Hence, there should be negligible impact towards the efficacy of antibody‐based therapies and vaccines that are currently being developed.

Methods

Ethical approval

Written informed consent was obtained from participants in accordance with the tenets of the Declaration of Helsinki. The study design protocol was approved by National Healthcare Group (NHG) Domain Specific Review Board (DSRB) under study number 2012/00917. Specimens from healthy donors were collected under study numbers 2017/2806 and NUS IRB 04‐140.

COVID‐19 patients and sample collection

Fifty‐seven patients who tested PCR‐positive for SARS‐CoV‐2 in nasopharyngeal swabs in Singapore were recruited into the study from January to March 2020 30 , 31 (Table 1). Patients were categorised into three groups based on clinical severity during hospitalisation: mild (no pneumonia on chest radiographs (CXR), n = 25), moderate (pneumonia on CXR without hypoxia, n = 19) and severe (pneumonia on CXR with hypoxia (desaturation to ≤ 94%), n = 13). Whole blood of patients was collected in BD Vacutainer® CPT™ tubes (BD Biosciences, Franklin Lakes, NJ, USA) and centrifuged at 1700 g for 20 min to obtain plasma fractions. Plasma samples were either heat‐inactivated at 56°C for 30 min, 17 or treated with Triton™ X‐100 (Thermo Fisher Scientific, Waltham, MA, USA) to a final concentration of 1% for 2 h at room temperature (RT) for virus inactivation. 31 , 32

Determining D614G mutation status of COVID‐19 patients

Residual clinical RNA was subjected to tiled amplicon PCR using ARTIC nCoV‐2019 version 3 panel. 33 Sequencing libraries were prepared using the Nextera XT and sequenced on MiSeq (Illumina, San Diego, California, USA) to generate 300 bp paired‐end reads. The reads were subjected to a hard‐trim of 50 bp on each side to remove primer artefacts using BBMap 34 prior to consensus sequence generation by Burrows‐Wheeler Aligner‐MEM v0.7.17. Sequences with nucleotide mutation A23403G were assigned as D614G.

Cells

Human embryonic kidney (HEK) 293T (ATCC, Manassas, VA, USA) cells were maintained in DMEM (Cytiva Life Sciences, Marlborough, MA USA) with 10% heat‐inactivated foetal bovine serum (FBS; Cytiva Life Sciences). CHO cells expressing human ACE2 (CHO‐ACE2; kindly gifted by Professor Yee‐Joo Tan, Department of Microbiology, NUS & IMCB, A*STAR, Singapore) were cultured in DMEM with 10% FBS, 1% MEM non‐essential amino acid solution (Thermo Fisher Scientific), and 0.5 mg mL‐1 of Geneticin selective antibiotic (Thermo Fisher Scientific). Surface expression of ACE2 on CHO‐ACE2 cells was confirmed using anti‐human ACE2 Alexa Fluor 647 (Santa Cruz Biotechnology, Dallas, TX, USA). All cells were maintained at 37°C with 5% CO2.

S‐flow assay

Full‐length SARS‐CoV‐2 Spike (S) protein of the D614 variant‐expressing HEK293T cells was produced by transduction with lentiviral particles. 35 Cells were seeded at 1.5 × 105 per well in 96‐well plates and incubated with Triton™ X‐100 inactivated plasma samples (1:100 dilution) in 10% FBS in PBS (FACS blocking buffer), followed by a secondary incubation of Alexa Fluor 647‐conjugated anti‐human IgM or IgG (1:500 dilution; Thermo Fisher Scientific) and propidium iodide (1:2500 dilution; Sigma‐Aldrich, St. Louis, MO, USA). Cells were acquired on BDTM LSR II laser (BD Biosciences), and results were analysed with FlowJo (version 10, Tree Star Inc. Becton Dickinson, Ashland, OR). Results are presented as percentage of binding, which indicates the percentage of cells with antibody binding.

SARS‐CoV‐2 pseudovirus production

The pseudotyped lentiviruses were produced as previously described. 3 Briefly, using the third‐generation lentivirus system, pseudotyped viral particles expressing SARS‐CoV‐2 D614 strain or G614 variant S proteins were generated by reverse transfection of 3 × 107 of HEK293T cells with 12 μg pMDLg/PRRE (Addgene, Watertown, Massachusetts, USA), 6 μg pRSV‐Rev (Addgene), 12 μg pTT5LnX‐coV‐SP (SARS‐CoV‐2 wildtype S, a kind gift from Dr Brendon John Hanson, DSO National Laboratories, Singapore) or pTT5Lnx‐coV‐SP‐D614G (SARS‐CoV‐2 mutant D614G S), and 24 μg pHIV‐Luc‐ZsGreen (Addgen) using Lipofectamine 2000 transfection (Invitrogen, Carlsbad, California, USA). Cells were cultured for 3 days, before viral supernatant was harvested by centrifugation to remove cell debris and filtered through a 0.45 μm filter unit (Sartorius, Gottingen, Germany). Viral titres were quantified with Lenti‐X™ p24 Rapid Titre Kit (Takara Bio, Kusatsu, Shiga, Japan).

Pseudovirus neutralisation assay

The pseudotyped lentivirus neutralisation assay was performed as previously described, with slight modifications. 3 CHO‐ACE2 cells were seeded at 3.2 x 104 per well in a 96‐well black microplate (Corning, New York, NY) in culture medium without Geneticin. Serially diluted heat‐inactivated plasma samples (1:10 to 1:31 250 dilutions) were incubated with equal volume of pseudovirus expressing SARS‐CoV‐2 S proteins of either original wildtype or D614G mutant strain (0.4 ng μL−1 of p24) at 37°C for 1 h, before being added to pre‐seeded CHO‐ACE2 cells. Cells were refreshed with culture media after 1 h incubation. After 48 h, cells were washed with PBS and lysed with 1× Passive Lysis Buffer (Promega, Madison, Wisconsin, USA) with gentle shaking at 125 rpm for 30 min at 37°C. Luciferase activity was subsequently quantified with Luciferase Assay System (Promega) on a GloMax Luminometer (Promega).

Data and statistical analysis

Data were analysed using GraphPad Prism (version 8.4.3; GraphPad Software, San Diego, CA) and Microsoft Excel (version 16.39; Microsoft). The Wilcoxon signed‐rank test and the paired t‐test were carried out to compare the antibody and neutralisation profiles of COVID‐19 patients at median of 31 and 98 days’ post‐illness onset (pio). P‐values less than 0.05 are considered to be statistically significant.

Conflict of interest

All authors declare no conflicts.

Author contributions

Cheryl Lee: Data curation; Formal analysis; Investigation; Methodology; Validation; Writing‐original draft; Writing‐review & editing. Siti Naqiah Amrun: Data curation; Formal analysis; Investigation; Methodology; Validation; Writing‐review & editing. Rhonda Chee: Data curation; Formal analysis; Investigation; Methodology; Validation; Writing‐review & editing. Yun Shan Goh: Data curation; Formal analysis; Investigation; Methodology; Writing‐review & editing. Tze‐Minn Mak: Data curation; Formal analysis; Investigation; Methodology; Writing‐review & editing. Sophie Octavia: Data curation; Formal analysis; Investigation; Methodology; Writing‐review & editing. Nicholas Yeo: Data curation; Formal analysis; Investigation; Methodology; Validation; Writing‐review & editing. Ziwei Chang: Data curation; Investigation; Methodology; Writing‐review & editing. Matthew Tay: Data curation; Investigation; Methodology; Writing‐review & editing. Anthony Torres‐Ruesta: Data curation; Formal analysis; Investigation; Methodology; Validation; Writing‐review & editing. Guillaume Carissimo: Formal analysis; Validation; Writing‐review & editing. Chek Meng Poh: Data curation; Investigation; Methodology; Writing‐review & editing. Siew‐Wai Fong: Formal analysis; Validation; Writing‐review & editing. Bei Wang: Resources; Supervision; Validation; Writing‐review & editing. Sandy Lee: Methodology; Validation; Writing‐review & editing. Barnaby Edward Young: Resources; Supervision; Validation; Writing‐review & editing. Seow‐Yen Tan: Resources; Supervision; Validation; Writing‐review & editing. Yee Sin Leo: Resources; Supervision; Validation; Writing‐review & editing. David Chien Lye: Resources; Supervision; Validation; Writing‐review & editing. Raymond Lin: Resources; Supervision; Validation; Writing‐review & editing. Sebastian Maurer‐Stroh: Data curation; Formal analysis; Investigation; Validation; Writing‐review & editing. Bernett Lee: Data curation; Formal analysis; Validation; Writing‐review & editing. Cheng‐I Wang: Resources; Supervision; Writing‐review & editing. Laurent Renia: Conceptualization; Methodology; Project administration; Supervision; Writing‐review & editing. Lisa FP Ng: Conceptualization; Funding acquisition; Methodology; Project administration; Supervision; Writing‐review & editing.

Supporting information

  

Click here for additional data file. (2.3MB, pdf)

Acknowledgments

The authors thank the study participants who donated their blood samples to this project and the healthcare workers caring for the COVID‐19 patients. The authors also wish to thank Ding Ying and the Singapore Infectious Disease Clinical Research Network (SCRN) for their help in patient recruitment and the staffs at the National Centre for Infectious Diseases (NCID) who assisted with data analysis on viral sequences and determination of the D614G status. The authors also thank Professor Yee‐Joo Tan (Department of Microbiology, NUS; Institute of Molecular and Cell Biology (IMCB), A*STAR) for kindly providing the CHO‐ACE2 cells and Dr Brendon John Hanson (DSO National Laboratories, Singapore) for kindly providing the SARS‐CoV‐2 wildtype S protein. This study was supported by core and COVID‐19 (H20/04/g1/006) research grants from Biomedical Research Council (BMRC) and the A*ccelerate GAP‐funded project (ACCL/20‐GAP001‐C20H‐E) from Agency for Science, Technology and Research (A*STAR), and National Medical Research Council (NMRC) COVID‐19 Research fund (COVID19RF‐001, COVID19RF‐007 and COVID19RF‐060). ATR is supported by the Singapore International Graduate Award (SINGA), A*STAR. The funding sources had no role in the study design; collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Contributor Information

Laurent Renia, Email: renia_laurent@immunol.a-star.edu.sg.

Lisa FP Ng, Email: lisa_ng@immunol.a-star.edu.sg.

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