key: cord-1035611-85n4hxj8
authors: Lee, Cheryl Yi‐Pin; Amrun, Siti Naqiah; Chee, Rhonda Sin‐Ling; Goh, Yun Shan; Mak, Tze‐Minn; Octavia, Sophie; Yeo, Nicholas Kim‐Wah; Chang, Zi Wei; Tay, Matthew Zirui; Torres‐Ruesta, Anthony; Carissimo, Guillaume; Poh, Chek Meng; Fong, Siew‐Wai; Bei, Wang; Lee, Sandy; Young, Barnaby Edward; Tan, Seow‐Yen; Leo, Yee‐Sin; Lye, David C; Lin, Raymond TP; Maurer‐Stroh, Sebastien; Lee, Bernett; Wang, Cheng‐I; Renia, Laurent; Ng, Lisa FP
title: Human neutralising antibodies elicited by SARS‐CoV‐2 non‐D614G variants offer cross‐protection against the SARS‐CoV‐2 D614G variant
date: 2021-02-22
journal: Clin Transl Immunology
DOI: 10.1002/cti2.1241
sha: 925dcc90a1a5f3a4690384d84abd0c3c3b18c167
doc_id: 1035611
cord_uid: 85n4hxj8

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.

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 immunebased interventions.

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 EC 50 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 

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 crossreactivity is essential for immunosurveillance, as well as to identify broadly neutralising antibodies or epitopes. 26 Here, we confirm that crossreactivity 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 . 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. Log 10 neutralisation EC 50 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 ttest (***P < 0.001). (f) Comparison of log 10 neutralisation EC 50 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).

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.

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 

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 â CPT TM 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 TM 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. 

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. 

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 9 10 7 of HEK293T cells with 12 lg pMDLg/PRRE (Addgene, Watertown, Massachusetts, USA), 6 lg pRSV-Rev (Addgene), 12 lg 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 lg 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 lm filter unit (Sartorius, Gottingen, Germany). Viral titres were quantified with Lenti-X TM p24 Rapid Titre Kit (Takara Bio, Kusatsu, Shiga, Japan).

The pseudotyped lentivirus neutralisation assay was performed as previously described, with slight modifications. 3 

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.

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.

Data curation; Formal analysis; Investigation

Methodology; Validation; Writing-review & editing. Ziwei Chang: Data curation; Investigation; Methodology; Writingreview & editing. Matthew Tay: Data curation

Investigation; Methodology; Writing-review & editing

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

Formal analysis; Validation; Writing-review & editing. Bei Wang: Resources; Supervision; Validation

Writing-review & editing. Sandy Lee: Methodology

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; Writingreview & editing. Raymond Lin: Resources; Supervision

Validation; Writing-review & editing. Sebastian Maurer-Stroh: Data curation; Formal analysis; Investigation

Bernett Lee: Data curation; Formal analysis; Validation; Writing-review & editing. Cheng-I Wang: Resources; Supervision; Writingreview & editing. Laurent Renia: Conceptualization

Project administration; Supervision; Writingreview & editing. Lisa FP Ng: Conceptualization; Funding acquisition

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All authors declare no conflicts. 

Additional supporting information may be found online in the Supporting Information section at the end of the article.This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.