Abstract
SARS-CoV-2 Spike amino acid replacements in the receptor binding domain (RBD) occur relatively frequently and some have a consequence for immune recognition. Here we report recurrent emergence and significant onward transmission of a six nucleotide deletion in the Spike gene, which results in loss of two amino acids: ΔH69/ΔV70. Of particular note this deletion often co-occurs with the receptor binding motif amino acid replacements N501Y, N439K and Y453F. In addition, we report a sub-lineage of over 350 sequences bearing seven spike mutations across the RBD (N501Y, A570D), S1 (ΔH69/V70) and S2 (P681H, T716I, S982A and D1118H) in England. Some of these mutations have possibly arisen as a result of the virus evolving from immune selection pressure in infected individuals. Enhanced surveillance for the ΔH69/ΔV70 deletion with and without RBD mutations should be considered as a priority.
Background
SARS-CoV-2’s Spike surface glycoprotein engagement of ACE2 is essential for virus entry and infection1, and the receptor is found in respiratory and gastrointestinal tracts2. Despite this critical interaction and related mutational constraints, it appears the RBD can tolerate mutations in this region3,4, raising the real possibility of virus escape from vaccines and monoclonal antibodies. Spike mutants exhibiting reduced susceptibility to monoclonal antibodies have been identified in in vitro screens5,6. Some of these have been found in clinical isolates7. The unprecedented scale of whole genome SARS-CoV-2 sequencing has enabled identification and epidemiological analysis of transmission. As of December 11th’ there were 246,534 SARS-CoV-2 sequences available in the GISAID initiative (https:gisaid.org/).
We recently documented de novo emergence of antibody escape mediated by Spike in an individual treated with convalescent plasma (CP), on the background of D614G8. Similarly, deletions in the NTD have been reported to provide escape for N-Terminal Domain-specific neutralising antibodies11. Dynamic changes in prevalence of Spike variants ΔH69/ΔV70 (an out of frame deletion) and D796H variant followed repeated use of CP, and in vitro the mutant displayed reduced susceptibility to the CP and multiple other sera, whilst retaining infectivity comparable to wild type8. We hypothesised that Spike ΔH69/ΔV70 arises either as a compensatory change and/or antibody evasion mechanism as suggested for other NTD deletions1, and therefore aimed to characterise specific circumstances around emergence of ΔH69/ΔV70 globally. Here we analysed the publicly available GISAID data for circulating SARS-CoV-2 sequences containing ΔH69/ΔV70.
Results
A deletion H69/V70 was present in over 3,000 sequences worldwide (2.5% of the available data) (Figure 1), and largely in Europe from where most of the sequences in GISAID are derived (Table 1). Many are from the UK and Denmark where sequencing rates are high compared to other countries. ΔH69/ΔV70 is observed in multiple different lineages, representing at least five independent acquisitions of the SARS-CoV-2 Spike ΔH69/ΔV70 deletion (Figure 1). The earliest samples that include the ΔH69/ΔV70 were detected in Thailand and Germany in January and February 2020, respectively, and are independent deletion events. The prevalence has since increased in other countries since August 2020 (Table 1). Further analysis of sequences revealed firstly that single deletions of either 69 or 70 were uncommon and secondly that some lineages of ΔH69/ΔV70 alone were present, as well as ΔH69/ΔV70 in the context of other mutations in Spike, specifically those in the RBD (Figure 1).
All sequences carrying the double-deletion were downloaded from the GISAID database and aligned to the Wuhan-Hu-1 reference strain using MAFFT. A series of global background sequences were also downloaded and used to place mutants into context. All duplicate sequences were removed. The inferred phylogeny suggests that there are multiple lineages of sequences carrying the ΔH69/V70, by itself (red), as well as with RDB mutations N501Y (dark blue), N453F (cyan) and Y439K (green).
All sequences containing the ΔH69/V70 deletion were extracted from the GISAID database (Accessed 26th Nov 2020) and tabulated according to both reporting country of origin and date in which they were posted online. The lineages carrying ΔH69/V70 began to expand in both Denmark and England in August 2020.
The structural impact of the double deletion was predicted by homology modelling of the spike NTD possessing ΔH69/ΔV70 using SWISS-MODEL. The ΔH69/ΔV70 deletion was predicted to alter the conformation of a protruding loop comprising residues 69-76, with the loop being predicted to be pulled in towards the NTD (Fig 2A). In the pre- and post-deletion conformations, the positions of the alpha carbons of residues 67 and 68 are roughly equivalent whereas the position of Ser71 in the post-deletion structure is estimated to have moved by approximately 6.7Å to approximately occupy the position of His69 in the pre-deletion structure. Concurrently, the positions of Gly72, Thr73, Asn74 and Gly75 are predicted to have changed by 6.5Å, 6.5Å, 4.7Å and 1.9Å respectively, with the overall effect of drawing these residues inwards, resulting in a less dramatically protruding loop; the position of Thr76 in the post-deletion model is roughly equivalent to its position in the pre-deletion structure.
A) Prediction of conformational change in the spike N-terminal domain due to deletion of residues His69 and Val70. The pre-deletion structure is shown in cyan, except for residues 69 and 70, which are shown in red. The predicted post-deletion structure is shown in green. Residues 66-77 of the pre-deletion structure are shown in stick representation and coloured by atom (carbon in cyan, nitrogen in blue, oxygen in coral). Yellow lines connect aligned residues 66-77 of the pre- and post-deletion structures and the distance of 6.5 Å between aligned alpha carbons of Thr73 in the pre- and post-deletion conformation is labelled. B) Surface representation of spike homotrimer in closed conformation (PDB: 6ZGE, Wrobel et al., 2020) homotrimer viewed in a ‘top-down’ view along the trimer axis with each monomer in shown in different shades of grey and locations of RBD mutations at residues 439, 453 and 501 highlighted in red. C) Spike in open conformation with a single erect RBD (PDB: 6ZGG, Wrobel et al. 2020) in trimer axis vertical view with the locations of deleted residues His69 and Val70 in the N-terminal domain and RBD mutations highlighted as red spheres and labelled on the monomer with erect RBD. Residues 71-75, which form the exposed loop undergoing conformational change in A, are omitted from this structure.
We next examined the lineages where Spike mutations in the RBD were identified at high frequency, in particular co-occurring with N439K, an amino acid replacement recently reported to be expanding in Europe and detected across the world3 (Figure 3, Supplementary figure 1). N439K appears to have reduced susceptibility to a small subset of monoclonals targeting the RBD, whilst retaining affinity for ACE2 in vitro3. The proportion of viruses with ΔH69/ΔV70 only increased from August 2020 when it co-occurred with the second N439K lineage3 (Figure 3). As of November 26th, remarkably there were twice as many cumulative sequences with the deletion as compared to the single N439K (Figure 3). Due to their high sampling rates the country with the highest proportion of N439K+ ΔH69/ΔV70 versus N439K alone is England. The low levels of sequencing in most countries indicate N439K’s prevalence could be relatively high3. In Scotland, where early growth of N439K was high (forming N439K lineage i that subsequently went extinct with other lineages after the lockdown3), there is now an inverse relationship with 546 versus 177 sequences for N439K and N439K+ΔH69/ΔV70 respectively (as of November 26th). These differences therefore likely reflect differing epidemic growth characteristics and timings of the introductions the N439K variants with or without the deletion.
All sequences in the GISAID database containing S:439K (3820 sequences, 26th November 2020) were realigned to Wuhan-Hu-1 using MAFFT. Viruses carrying the Spike double deletion ΔH69/V70 (red) emerged and expanded from viruses with S:439K (black).
Between August and October 2020, an exponential increase in mutants carrying both 439K and ΔH69/V70 saw the latter become dominant in terms of cumulative cases.
The second significant cluster with ΔH69/ΔV70 and RBD mutants involves Y453F, another RBD mutation that increases binding affinity to ACE2, along with F486L and N501T related to human-mink transmissions in Denmark9 (Figure 4). This sub-lineage, termed ‘Cluster 5’ was part of a wider lineage in which the same deleted region (ΔH69/ΔV70) was observed. In Y453F lineages, the mutant virus demonstrates reduced susceptibility to sera from recovered COVID-19 patients (https://files.ssi.dk/Mink-cluster-5-short-report_AFO2). The ΔH69/ΔV70 was first detected in the Y453F background on August 24th and thus far appears limited to Danish sequences.
All 753 publicly available Mink origin Spike sequences were downloaded from the GISAID database (accessed 12th December) and aligned to the Wuhan-Hu-1 reference sequence using MAFFT. Acquisition of the Spike mutant Y453F, an exposed region in the RBD, occurred early on in Dutch Mink (green circles). Later in infection, 453F was acquired by Danish Mink (red) and subsequently, those with the 453F mutant also acquired the ΔH69/V70 double deletion (purple).
A third lineage containing the same out of frame deletion ΔH69/ΔV70 has arisen with another RBD mutation N501Y (Figure 5, supplementary figure 2). Based on its location it might be expected to escape antibodies similar to COV2-24995. In addition, when SARS-CoV-2 was passaged in mice for adaptation purposes, N501Y emerged and increased pathogenicity. Early sequences with N501Y alone were isolated both in Brazil and USA in April 2020. N501Y + ΔH69/ΔV70sequences appear to have been detected first in the UK in September 2020, with the crude cumulative number of N501Y + ΔH69/ΔV70 mutated sequences now exceeding the single mutant (Figure 5). Of particular concern is a sublineage of around 350 sequences (Figure 6) bearing six spike mutations across the RBD (N501Y, A570D) and S2 (P681H, T716I, S982A and D1118H) as well as the ΔH69/ΔV70 in England (Figure 7). This cluster has a very long branch (Figure 6).
All sequences in the GISAID database containing S:501Y were downloaded and realigned to Wuhan-Hu-1 using MAFFT. Sequences were broadly split into four major clades; sequences carrying the Spike double deletion ΔH69/V70 (red) formed an entirely separate clade from non-carriers. Sequences carrying 501Y but an absence of ΔH69/V70 formed a second lineage and appeared to expand only in Wales (green). Another major clade (blue) was limited entirely to Australia and finally a fourth clade (black) was limited to several African countries and Brazil.
Between October and November 2020, the sequences carrying both 501Y and the ΔH69/V70 also became dominant.
Global maximum likelihood phylogeny of all ΔH69/V70 sequences downloaded from the GISAID database (3066 sequences, accessed 26th November 2020). A distinct sub-lineage of ΔH69/V70 sequences (red) developed with six linked S mutations, two of which are in the RBD (N501Y and A570D) and four others in S2 (P681H, T716I, S982A and D1118H). All five mutations occur together in all cases with the deletion.
Spike homotrimer in open conformation with one upright RBD (PDB: 6ZGE, Wrobel et al., 2020) with different monomers shown in shades of grey. To the left, surface representation overlaid with ribbon representation and to the right, opaque surface representation accentuating the locations of surface-exposed residues. The deleted residues 69 and 70 and the residues involved in amino acid substitutions (501, 570, 716, 982 and 1118) are coloured red. The location of an exposed loop including residue 681 is absent from the structure, though the residues either side of the unmodelled residues, 676 and 689, are coloured orange. On the left structure, highlighted residues are labelled on the monomer with an upright RBD; on the right structure, all visible highlighted residues are labelled.
Discussion
We have presented data demonstrating multiple, independent, and circulating lineages of SARS-CoV-2 variants bearing a Spike ΔH69/ΔV70. This deletion spanning six nucleotides, is mostly due to an out of frame deletion of six nucleotides, has frequently followed receptor binding amino acid replacements (N501Y, N439K and Y453F that have been shown to reduce binding with monoclonal antibodies) and its prevalence is rising in parts of Europe, with the greatest increases since August 2020.
A recent analysis highlighted the potential for enhanced transmissibility of viruses with deletions in the N terminal domain, including ΔH69/ΔV7011. The potential for SARS-CoV-2 mutations to rapidly emerge and fix is exemplified by D614G, an amino acid replacement in S2 that alters linkages between S1 and S2 subunits on adjacent protomers as well as RBD orientation, infectivity, and transmission12–14. The example of D614G also demonstrates that mechanisms directly impacting important biological processes can be indirect. Similarly, a number of possible mechanistic explanations may underlie ΔH69/ΔV70. For example, the fact that it sits on an exposed surface might be suggestive of immune interactions and escape, although allosteric interactions could alternatively lead to higher infectivity.
The finding of a sub-lineage of over 350 sequences bearing seven spike mutations across the RBD (N501Y, A570D), S1 (ΔH69/ΔV70) and S2 (P681H, T716I, S982A and D1118H) in England requires careful monitoring. The detection of a high number of novel mutations suggests this lineage has either been introduced from a geographic region with very poor sampling or viral evolution may have occurred in a single individual in the context of a chronic infection8.
Given the emergence of multiple clusters of variants carrying RBD mutations and the ΔH69/ΔV70 deletion, limitation of transmission takes on a renewed urgency. Concerted global vaccination efforts with wide coverage should be accelerated. Continued emphasis on testing/tracing, social distancing and mask wearing are essential, with investment in other novel methods to limit transmission15. Detection of the deletion by rapid diagnostics should be a research priority as such tests could be used as a proxy for antibody escape mutations to inform surveillance at global scale.
Conflicts of interest
RKG has received consulting fees from UMOVIS lab, Gilead Sciences and ViiV Healthcare, and a research grant from InvisiSmart Technologies.
Methods
Phylogenetic Analysis
All available full-genome SARS-CoV-2 sequences were downloaded from the GISAID database (http://gisaid.org/) 16 on 26th November. Duplicate and low-quality sequences (>5% N regions) were removed, leaving a dataset of 194,265 sequences with a length of >29,000bp. All sequences were realigned to the SARS-CoV-2 reference strain MN908947.3, using MAFFT v7.473 with automatic flavour selection and the --keeplength --addfragments options17. Major SARS-CoV-2 clade memberships were assigned to all sequences using the Nextclade server v0.9 (https://clades.nextstrain.org/).
Maximum likelihood phylogenetic trees were produced using the above curated dataset using IQ-TREE v2.1.2 18. Evolutionary model selection for trees were inferred using ModelFinder 19 and trees were estimated using the GTR+F+I model with 1000 ultrafast bootstrap replicates20. All trees were visualised with Figtree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/), rooted on the SARS-CoV-2 reference sequence and nodes arranged in descending order. Nodes with bootstraps values of <50 were collapsed using an in-house script.
Pseudotype virus preparation
Viral vectors were prepared by transfection of 293T cells by using Fugene HD transfection reagent (Promega). 293T cells were transfected with a mixture of 11ul of Fugene HD, 1μg of pCDNAΔ19Spike-HA, 1ug of p8.91 HIV-1 gag-pol expression vector22,23, and 1.5μg of pCSFLW (expressing the firefly luciferase reporter gene with the HIV-1 packaging signal). Viral supernatant was collected at 48 and 72h after transfection, filtered through 0.45um filter and stored at −80°C as previously described24. Infectivity was measured by luciferase detection in target TZMBL transduced to express TMPRSS2 and ACE2.
Normalisation of virus titre by SG-PERT to measure RT activity in lentivirus preparation
Supernatant was subjected to SG-PERT as previously described.25
Homology modelling
Prediction of conformational change in the spike N-terminal assessed by homology modelling of the NTD (residues 14-306) predicted by homology modelling using SWISS-MODEL26 with template chain A of PDB 7C2L27 and aligned with 7C2L using PyMOL. Figures prepared with PyMOL (Schrödinger) using PDBs 7C2L, 6ZGE28 and 6ZGG28.
Acknowledgements
RKG is supported by a Wellcome Trust Senior Fellowship in Clinical Science (WT108082AIA). DLR is funded by the MRC (MC UU 1201412). WH is funded by the MRC (MR/R024758/1). We thank Dr James Voss for the kind gift of HeLa cells stably expressing ACE2.
Footnotes
Author affiliation has been corrected
