key: cord-0780313-xf7mmuj1
authors: Ruiz-Rodriguez, P.; Frances-Gomez, C.; Chiner-Oms, A.; Lampoacutepez, M. G.; Jimampeacutenez-Serrano, S.; Cancino-Muampntildeoz, I.; Ruiz-Hueso, P.; Torres-Puente, M.; Bracho, M. A.; D'Auria, G.; Martinez-Priego, L.; Guerreiro, M.; Montero-Alonso, M.; Gomez, M. D.; Piampntildeana, J. L.; SeqCOVID-SPAIN consortium,; Gonzampaacutelez-Candelas, F.; Comas, I.; Marina, A.; Geller, R.; Coscolla, M.
title: Evolutionary and phenotypic characterization of spike mutations in a new SARS-CoV-2 Lineage reveals two Variants of Interest
date: 2021-03-12
journal: nan
DOI: 10.1101/2021.03.08.21253075
sha: c142830ff107aa1b9dc518b83f30edca1353d990
doc_id: 780313
cord_uid: xf7mmuj1

Molecular epidemiology of SARS-CoV-2 aims to monitor the appearance of new variants with the potential to change the virulence or transmissibility of the virus. During the first year of SARS-CoV-2 evolution, numerous variants with possible public health impact have emerged. We have detected two mutations in the Spike protein at amino acid positions 1163 and 1167 that have appeared independently multiple times in different genetic backgrounds, indicating they may increase viral fitness. Interestingly, the majority of these sequences appear in transmission clusters, with the genotype encoding mutations at both positions increasing in frequency more than single-site mutants. This genetic outcome that we denote as Lineage B.1.177.637, belongs to clade 20E and includes 12 additional single nucleotide polymorphisms but no deletions with respect to the reference genome (first sequence in Wuhan). B.1.177.637 appeared after the first wave of the epidemic in Spain, and subsequently spread to eight additional countries, increasing in frequency among sequences in public databases. Positions 1163 and 1167 of the Spike protein are situated in the HR2 domain, which is implicated in the fusion of the host and viral membranes. To better understand the effect of these mutations on the virus, we examined whether B.1.177.637 altered infectivity, thermal stability, or antibody sensitivity. Unexpectedly, we observed reduced infectivity of this variant relative to the ancestral 20E variant in vitro while the levels of viral RNA in nasopharyngeal swabs did not vary significantly. In addition, we found the mutations do not impact thermal stability or antibody susceptibility in vaccinated individuals but display a moderate reduction in sensitivity to neutralization by convalescent sera from early stages of the pandemic. Altogether, this lineage could be considered a Variant of Interest (VOI), we denote VOI1163.7. Finally, we detected a sub-cluster of sequences within VOI1163.7 that have acquired two additional changes previously associated with antibody escape and it could be identified as VOI1163.7.V2. Overall, we have detected the spread of a new Spike variant that may be advantageous to the virus and whose continuous transmission poses risks by the acquisition of additional mutations that could affect pre-existing immunity.

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The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.08.21253075 doi: medRxiv preprint Introduction 52 Genomic surveillance of viral mutations is the first step in detecting viral changes that could 53 impact public health by interfering with diagnostics, modifying pathogenicity, or altering 54 susceptibility to existing immunity or treatments. In many countries, the challenge of detecting 55 new mutations of interest in SARS-CoV-2 is approached by sequencing representative genomes 56 from circulating viruses, sharing sequence information on public databases (e.g. GISAID 1 ), and 57 analysing them in real-time using platforms, such as Nextstrain 2 . While mutations appear 58 randomly, their fate in the population depends on a combination of the conferred fitness 59 advantage as well as stochastic and demographic processes. A first step in assessing the 60 potential public health impact of mutations is to decipher if their increase in frequency is due to 61 chance or adaptation. If found to be adaptive, it is important to evaluate whether their 62 adaptation is linked to an improved ability to replicate, colonize, transmit, or evade antiviral 63 hosts defences 3 . An important challenge in the field is to decipher which of all the variants that 64 appear should be monitored to implement measures to mitigate their risk to public health. 65

Genotypes that are phenotypically different from a reference isolate or have mutations that lead 66 to changes associated with either established or suspected phenotypes could be considered 67

Variants of Interest (VOI) if they also fit one of the following criteria: i) cause community 68 transmission/multiple COVID-19 clusters or ii) have been detected in multiple countries 4 . Among 69 VOI, only those genotypes that are associated with higher transmissibility, with detrimental 70 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.08.21253075 doi: medRxiv preprint 9 England. However, after three months, cluster 1163.654 is no longer being detected. A large 181 cluster within Lineage 20E is formed by 37 sequences with the mutation G1167F. Sequences for 182 this cluster were obtained in Wales between the end of October and the beginning of November 183 2020 (dark pink in the external circle in Supplementary Fig.2g) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.08.21253075 doi: medRxiv preprint including four transmission clusters and nine unique sequences ( Supplementary Fig.4) , while 206 mutations at G1167 were observed in at least five independent sequences. Interestingly, 207 D1163Y and G1167V were observed together in only one individual within 20I/501Y.V1, 208 although they were not fixed (relative frequency of 27% and 17% of the reads with D1163Y and 209 G1167V, respectively; Supplementary table 3) . Finally, only two sequences that harbour the 210 amino acid replacement G1167V in the S protein were observed in the genomic background 211 20H/501Y.V2 (Supplementary table 3) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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The copyright holder for this this version posted March 12, 2021. reported 19,21,51 , the 20E S genotype enhanced infectivity relative to the Wuhan S genotype by 306 70% in both Vero (p-value = 0.005 by unpaired t-test; Figure 3a ) and A549-hACE2-TMPRSS2 cells 307 (p-value = 0.016 by unpaired t-test; Figure 3b ). The 20E S genotype also showed a trend towards 308 increased infectivity versus the D614G mutation alone (35% increase in both cell lines), as has 309 been previously reported 49 , yet the difference was not statistically significant (p-value > 0.05 by 310 unpaired t-test; Figure 3a is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint (A222V and D614G) , where p-value = 0.01; Figure 3d) is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.08.21253075 doi: medRxiv preprint wave (ID80; mean = 1.03, range: 0.87-1.23; p-value = 0.83 by paired t-test; Figure 4b ). These 369 results indicate that the D1163Y and G1167V mutations can provide some degree of escape from 370 pre-existing antibody-based immunity relative to the 20E S genotype depending on the genomic 371 background of the infecting genotype. As a modest reduction in titers was observed with sera 372 from early in the pandemic (Figure 4a ), which is more closely related to the current S genotype 373 present in approved vaccines 55,56 , we examined if B.1.177.637 S genotype resulted in reduced 374 neutralization by sera from donors vaccinated with the BNT162b2 vaccine. No significant 375 differences in susceptibility to antibody neutralization from vaccinated donors were observed 376 between the two genotypes, indicating that VOI1163.7 is unlikely to alter the efficacy of vaccines 377 based on the Wuhan S genotype (Figure 4c) . 378

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The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.08.21253075 doi: medRxiv preprint importance, as they can significantly impact public health. In this work, we have identified two 390 mutations in the S protein that are likely to be beneficial for the virus based on several lines of 391 evidence. First, these mutations are highly variable within SARS-CoV-2 but conserved across the 392 closely related coronaviruses. Second, the vast majority of sequences harbouring these 393 mutations appear in clusters (Figure 1a and 1b) . Third, the largest cluster, and therefore the 394 most successful in terms of transmission, includes both mutations together (Figure 1a and 1b) . 395

Additionally, both positions have been reported as positively selected multiple times throughout 396 the SARS-CoV-2 phylogeny indicating a fitness advantage 57 . Although either mutation in 397 isolation could be advantageous, their co-occurrence in a large cluster that has been sustained 398 for more than six months across Europe is suggestive of increased fitness when both mutations 399 are present together. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 12, 2021. ; https://doi.org/10.1101/2021.03.08.21253075 doi: medRxiv preprint 21 burial in the HR2 coiled-coil leucine zipper of the pre-fusion state (Figure 2 ). This extensive 405 flexibility of S prefusion stalk seems to be unique to the SARS-CoV-2 S protein and has not been 406 reported for other class I fusion proteins 18 . The stalk flexibility has been suggested to increase 407 avidity for the host receptors by allowing the engagement of multiple S proteins 18 . Therefore, 408 stalk stabilization is likely to result in a reduced ability of S to bind receptors in the target cell. 409

Indeed, we find the B.1.177.637 genotype to have reduced infectivity compared to the 20E 410 genotype in both Vero and A549-hACE2-TMPRSS2 cells (Figure 3a is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint eliciting T-cell responses in convalescent COVID-19 cases 60 as well as in SARS-CoV-2-naïve 433 individuals 53 , indicating cross-reactivity in epitopes involving these regions. B cell linear epitopes 434 that span D1163 and G1167 have also been reported 52 , with D1163 belonging to a dominant 435 linear B cell epitope recognized by more than 40% COVID-19 patients used in the assay 54 . D1163 436 is fully solvent exposed in available structure 18,45 , making its side-chain easily accessible to 437 antibodies, providing a potential mechanism for altering antibody binding. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Whether VOI1163.7.V1 and VOI1163.7.V2 will continue to increase in frequency and accumulate 473 additional mutations that could improve its fitness and/or present challenges to vaccines or 474 diagnostics remains to be seen. However, their characterization as VOI would help to discover if 475 enough evidence holds to consider them VOC and therefore required monitoring. 476 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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14 sequences including SARS-COV-2 belonging to sarbecoviruses, sequences were annotated 500 with annotation files available at NCBI database in order to locate the spike gene coordinates 501 (accession numbers are available at supplemental table 1) is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 12, 2021. Table 6 ). 537

The dataset to represent 20I/501Y.V1 phylogenetic relationships include 3,067 randomly 538 selected samples identified by Pangolin typing system (https://github.com/cov-539 lineages/pangolin) as lineage B.1.1.7 plus the 33 sequences with amino acid replacements in 540 D1163 and/or G1167 (Supplementary Table 7) . 541

For all the alignments, problematic positions reported by Lanfear, R. 77 were masked for the 542 phylogenetic reconstruction using masked_alignment.sh script. 543

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Maximum-likelihood phylogenies in Figure 1 and supplementary Supplementary Fig.2, S4 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The mean GFP signal observed in several mock-infected wells was subtracted from all infected 587 wells, followed by standardization of the GFP signal to the mean GFP signal from wells incubated 588 at 30.4°C. Finally, a three parameter log-logistic function was fitted to the data using the drc 589

package v 3.0-1 in R (LL.3 function) and the temperature resulting in 50% inhibition calculated 590 using the drc ED function. Statistical differences in the temperature resulting in 50% reduction 591 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Evaluation of neutralization by convalescent sera and efficacy of virus particle production. 595 Pseudotyped VSV bearing 20E or B.1.177.637 S variants were evaluated for sensitivity to 596 neutralization by convalescent sera as previously described 83 with slight modifications. Briefly, 597 16-hours post-infection, GFP signal in each well was determined using an Incucyte S3 (Essen 598 Biosciences). The mean GFP signal observed in several mock-infected wells was subtracted from 599 all infected wells, followed by standardization of the GFP signal in each well infected with 600 antibody-treated virus to that of the mean GFP signal from wells infected with mock-treated 601 virus. Any negative values resulting from background subtraction were arbitrarily assigned a low, 602 non-zero value (10 -5 ). The serum dilutions were then converted to their reciprocal, their 603 logarithm (Log10) was taken, and the dose resulting in 50% (ID50) or 80% (ID80) reduction in GFP 604 signal was calculated in R using the drc package v 3.0-1. A two-parameter log-logistic regression 605 (LL2 function) was used for all samples except when a three-parameter logistic regression 606 provided a significant improvement to fit, as judged by the ANOVA function in the drc package 607 (e.g. p < 0.05 following multiple testing correction using the Bonferroni method). The script for 608 calculating the ID50 and ID80 as well as the standardized GFP signal for each condition is 609 available at https://github.com/PathoGenOmics/B.1.177.637_SARS-CoV-2. For the first wave, 610 serum samples and data from patients included in this study were provided by the Consorcio 611

Hospital General de Valencia Biobank, integrated in the Valencian Biobanking Network, and they 612 were processed following standard operating procedures with the appropriate approval of the 613 Ethics and Scientific Committees. All first wave samples were obtained from donors that were 614 admitted to the intensive care unit and were collected during April 2020. For the second wave 615 donors, sera were obtained (October 2020) from severe COVID-19 patients requiring inpatient 616 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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