A Unique SARS-Cov-2 Spike Protein P681H Variant Detected in Israel

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Brief Report A Unique SARS-CoV-2 Spike Protein P681H Variant Detected in Israel

Neta S. Zuckerman 1,*, Shay Fleishon 1, Efrat Bucris 1, Dana Bar-Ilan 1 , Michal Linial 2 , Itay Bar-Or 1, Victoria Indenbaum 1, Merav Weil 1 , Yaniv Lustig 1, Ella Mendelson 1,3, Michal Mandelboim 1,3,†, Orna Mor 1,3,†, Neta Zuckerman 1 and on behalf of the Israel National Consortium for SARS-CoV-2 Sequencing ‡

1 Central Virology Laboratory, Israel Ministry of Health, Sheba Medical Center, Tel-Hashomer 52621, Israel; s.ﬂ[email protected] (S.F.); [email protected] (E.B.); [email protected] (D.B.-I.); [email protected] (I.B.-O.); [email protected] (V.I.); [email protected] (M.W.); [email protected] (Y.L.); [email protected] (E.M.); [email protected] (M.M.); [email protected] (O.M.); tozaot.negiﬁ[email protected] (N.Z.) 2 Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; [email protected] 3 Department of Epidemiology and Preventive Medicine, School of Public Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel * Correspondence: [email protected]; Tel.: +972-35-302-341 † These authors contributed equally to the manuscript. ‡ Israel National Consortium for SARS-CoV-2 sequencing: Neta Zuckerman, Efrat Dahan Bucris, Michal Mandelboim, Dana Bar-Ilan, Oran Erster, Tzvia Mann, Omer Murik, David A. Zeevi, Assaf Rokney, Joseph Jaffe, Eva Nachum, Maya Davidovich Cohen, Ephraim Fass, Gal Zizelski Valenci, Mor Rubinstein, Efrat Rorman, Israel Nissan, Efrat Glick-Saar, Omri Nayshool, Gideon Rechavi, Ella Mendelson, Orna Mor.

Citation: Zuckerman, N.S.; Fleishon, Abstract: The routine detection, surveillance, and reporting of novel SARS-CoV-2 variants is crucial, S.; Bucris, E.; Bar-Ilan, D.; Linial, M.; Bar-Or, I.; Indenbaum, V.; Weil, M.; as these threaten to hinder global vaccination efforts. Herein we report a novel local variant with a Lustig, Y.; Mendelson, E.; et al. A non-synonymous mutation in the spike (S) protein P681H. This local Israeli variant was not associated Unique SARS-CoV-2 Spike Protein with a higher infection rate or higher prevalence. Furthermore, the local variant was successfully P681H Variant Detected in Israel. neutralized by sera from fully vaccinated individuals at a comparable level to the B.1.1.7 variant and Vaccines 2021, 9, 616. https:// an Israel wild-type strain. While it is not a variant of concern, routine monitoring by sequencing is doi.org/10.3390/vaccines9060616 still required.

Academic Editors: Steven B. Bradfute, Keywords: SARS-CoV-2; sequencing; virus neutralization; Covid-19; variant of concern Ralph A. Tripp and Scott Anthony

Received: 19 April 2021 Accepted: 3 June 2021 1. Introduction Published: 8 June 2021 SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) emerged in Wuhan,

Publisher’s Note: MDPI stays neutral China in December 2019 [1] and has rapidly spread worldwide, causing severe respiratory with regard to jurisdictional claims in disease and leading to substantial morbidity and mortality. published maps and institutional affil- Global whole genome sequencing (WGS) efforts and initiatives, such as the GISAID iations. (global initiative on sharing all influenza data) platform [2], Nextstrain [3], and Pan- golin [4,5], facilitate access to worldwide SARS-CoV-2 sequences, tracking different lineages of the virus and detecting emerging novel virus variants. Recently, various variants of concern (VOC) have been identified globally, including the B.1.1.7 variant identified in the UK [6], the B.1.351 variant identified in South Africa [7], and the P.1 variant identified in Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Brazil [8]. The VOCs are fast spreading and characterized by higher infection rates or lower This article is an open access article neutralization in convalescent or vaccinated individuals, consistent with evolutionary distributed under the terms and advantages for these variants [9,10]. conditions of the Creative Commons Mutations in the spike (S) protein of SARS-CoV-2 attract much attention in monitoring Attribution (CC BY) license (https:// emerging strains, as infection is driven by the S protein binding the cell surface angiotensin- creativecommons.org/licenses/by/ converting enzyme 2 (ACE2) receptor on human cells [11]. Mutations in the SARS-CoV-2 4.0/). genome affecting the S protein have emerged in numerous differentially-reported variants,

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such as the N501Y mutation that is shared by the currently known VOCs, B.1.1.7, B.1.351, and P.1, and which increase the affinity of the S protein to its receptor binding domain (RBD) [12]. Similarly, position 681 in the S protein, located in the vicinity of a critical furin cleavage site, has been found to be mutated in several variants. S protein cleavage (after R685 residue) leads to S protein activation by separating the protein to S1 and S2 subunits. Following the RBD binding to the ACE2 receptor, the S1/S2 activation step leads to membrane fusion and the entering of the viral genome into human cells [13]. The P681H mutation was observed as early as March 2020 in samples worldwide, such as in Nigeria [3], Hawaii [4], and recently in three independent variants in New York [14], and also characterizes the globally spreading B.1.1.7 VOC [6]. It is postulated that the P681H mutation enhances the transmissibility of the virus by facilitating a conformational change in the S protein following protease activity at the cell membrane [15]. Additionally, the A.23.1 variant that has been reported as the dominant lineage in Uganda [16] and the B.1.617 family of variants identified in India (not published) acquired the P681R mutation in the same position. In this study, we report a novel variant that emerged from a locally circulating lineage, B.1.1.50. This variant is characterized by the non-synonymous S protein mutation P681H and four additional synonymous mutations. This B.1.1.50 + P681H variant was identified in clinical samples in Israel between November 2020 and January 2021, whereas the P681H mutation had already been identified in Israel via sequencing of SARS-CoV-2 positive sewage samples in October 2020. This novel variant has not been associated with a higher infection rate and its frequency has declined in correlation with the increase of the B.1.1.7 variant in Israel. Neutralization assays with sera from vaccinated individuals showed comparable neutralization levels to the B.1.1.7 variant and an Israel wild-type (WT) strain. With increasing reports on emerging VOC worldwide threatening to hinder global vaccination efforts, the routine surveillance and reporting of local variants, which have the potential for spreading globally, is extremely important in the battle against Covid-19 and further spreading of SARS-CoV-2.

2. Materials and Methods 2.1. Sample Collection for Sequencing Random sampling of SARS-CoV-2 PCR-positive respiratory samples collected via a convenient sampling design from major testing laboratories covering the majority of Israel has been conducted in Israel as part of the national effort for monitoring SARS-CoV-2 variants, starting in December 2020. Likewise, extracted nucleic acids from SARS-CoV-2 PCR- positive sewage samples collected for environmental surveillance of circulating viruses [17] were used for WGS.

2.2. Library Preparation and Sequencing Viral genomes were extracted from 200 µL respiratory samples with a MagNA PURE 96 (Roche, Germany), according to the manufacturer instructions. Libraries were prepared using a COVID-seq library preparation kit, as per manufacturer’s instructions (Illumina). Library validation and mean fragment size were determined using TapeStation 4200 via DNA HS D1000 kit (Agilent). Libraries were pooled, denatured, and diluted to 10 pM and sequenced on NovaSeq (Illumina, San Diego, CA, USA).

2.3. Bioinformatics Analysis Fastq files were subjected to quality control using FastQC (www.bioinformatics. babraham.ac.uk/projects/fastqc/, accessed on 15 February 2021) and MultiQC [18], and low-quality sequences were filtered using trimmomatic [19]. Sequences were mapped to the SARS-CoV-2 reference genomes (NC_045512.2) using the Burrows–Wheeler aligner (BWA) mem [20]. Resulting BAM files were sorted, indexed, and subjected to quality control using SAMtools suite [21]. Coverage and depth of sequencing was calculated from sorted BAM files using a custom python script. Consensus fasta sequences were assembled for each Vaccines 2021, 9, 616 3 of 9

sample using iVar (https://andersen-lab.github.io/ivar/html/index.html, accessed on 1 June 2021), with positions with <5 nucleotides determined as Ns. Multiple alignment of sample sequences with SARS-CoV-2 reference genome (NC_045512.2) was performed using MAFFT [22]. Mutation calling, translation to amino acid, and identification of P681H variant sequences were carried out in R with a custom code using the Bioconductor package Se- qinr [23]. Sequences were further analyzed together with additional sequences identified as belonging to the background lineage B.1.1.50 downloaded from GISAID [2]. Phylogenetic trees were constructed using the Augur pipeline [3]. Sequences were aligned to the SARS-CoV-2 reference genome (NC_045512.2) using MAFFT [22], and a time-resolved phylogenetic tree was constructed with IQ-Tree [24] and TreeTime [25] under the GTR substitution model and visualized with auspice [3]. Lineage nomenclature was attained from Pangolin Lineages [4]. Analysis of sewage samples was conducted using a custom R code and the Biocon- ductor package Rsamtools [26]. BAM files were imported to R and the frequency of each mutated position along the genome, out of the total number of nucleotides covering that position, was obtained. The frequency of the P681H mutation in the spike protein was recorded for each sample.

2.4. Neutralization Assays VERO-E6 cells at a concentration of 20*103/well were seeded in sterile 96-wells plates with 10% FCS MEM-EAGLE medium, and stored at 37 ◦C for 24 h. One hundred TCID50 of B.1.1.50 + P681H variant, a strain commonly circulating in Israel lacking a P681H mutation (Israel WT), and B.1.1.7 variant isolates were incubated with inactivated sera from fully vaccinated individuals diluted 1:10 to 1:1280 in 96 well plates for 60 min at 33 ◦C. Virus– serum mixtures were added to the VERO E6 cells and incubated for ﬁve days at 33 ◦C and stained with Gentain violet staining (1%) to identify the highest serum dilution having no viral cytopathic effect. A dilution equal to 1:10 or above was considered neutralizing.

3. Results 3.1. Characterization of the B.1.1.50 + P681H Variant Phylogenetic trees representing randomly chosen samples sequenced in Israel are routinely constructed as part of the national effort for SARS-CoV-2 sequencing and variant surveillance. Two distinct clusters, each with the P681H mutation, were observed; a large cluster, representing the B.1.1.7 variant and a separate smaller cluster emerging from the B.1.1.50 lineage with the P681H mutation (Figure1A). B.1.1.50 is a locally circulating lineage, in which the majority of the sequences are from Israel (70%), in addition to sequences from the Palestinian authorities (12%) and the UK (12%) [4]. The B.1.1.50 + P681H variant is characterized by the non-synonymous S protein mutation P681H (C23604A) and four additional synonymous mutations: Nsp3:C7765T, Nsp12b:C13821T, Nsp16:T21111C, and C29545A. The B.1.1.50 + P681H variant is unique to Israel, aside from 2 sequences originating in the Palestinian authorities, sharing a border with Israel. A sub-lineage of the variant contains an additional non-synonymous mutation in the S protein at position A27S (G21641T) (Figure1B). Up to January 2021, a total of 181 individuals were detected with the B.1.1.50 + P681H variant. Most of these individuals were randomly sampled from various districts across Israel, with no gender or age bias. The prevalence of the variant in the population decreased with time, from 12.7% out of 212 randomly sequenced sampled in November 2020, to 8.3% in December 2020 (out of 854 random samples), to 3.3% in January (out of 2522 random samples) (Table1). On the other hand, the B.1.1.7 variant, ﬁrst identiﬁed in Israel in December 2020 and now the dominant lineage in Israel, increased with time, from 0% in November to ~40% in January. Vaccines 2021, 9, x 4 of 9 Vaccines 2021, 9, 616 4 of 9

FigureFigure 1.1. CharacterizationCharacterization ofof thethe B.1.1.50B.1.1.50 ++ P681HP681H variant.variant. PhylogeneticPhylogenetic treestrees highlightinghighlighting thethe genotypegenotype atat sitesite 681681 inin thethe SS protein,protein, withwith thethe wild-typewild-type prolineproline (P)(P) inin greengreen andand thethe mutationsmutations histidinehistidine (H)(H) andand argininearginine (R)(R) inin yellowyellow andand blue,blue, respectively.respectively. ( A(A)) SARS-CoV-2 SARS-CoV-2 genomes genomes sequenced sequenced in in Israel Israel from from March March 2020 2020 to to January January 2021 2021 ( n(n= =2482). 2482). TheThe mainmain clustersclusters harboringharboring thethe P681HP681H mutationmutation (in(in yellow)yellow) areare the the B.1.1.7 B.1.1.7 (20I/501Y.V1) (20I/501Y.V1) andand thethe B.1.1.50B.1.1.50 ++ P681HP681H variant.variant. ((BB)) SARS-CoV-2SARS-CoV-2 genomesgenomes fromfrom ofof thethe B.1.1.50B.1.1.50 lineagelineage onlyonly ((nn= = 489).489). The B.1.1.50 ++ P681H cluster is co composedmposed of local (Israel) viruses only, with the exception of 2 sequences from isolates identified in the Palestinian authorities, and includes a sub-cluster with an with the exception of 2 sequences from isolates identified in the Palestinian authorities, and includes a sub-cluster with an additional S protein non-synonymous mutation, A27S. Additional mutations are listed by each branch. Phylogenetic trees additional S protein non-synonymous mutation, A27S. Additional mutations are listed by each branch. Phylogenetic trees were created with Nextstrain Augur pipeline and visualized with Auspice [3]. Non-Israeli B.1.1.50 lineage sequences were weredownloaded created withfrom Nextstrain GISAID and Augur identified pipeline with and Pangolin visualized classification with Auspice [4]. [3]. Non-Israeli B.1.1.50 lineage sequences were downloaded from GISAID and identified with Pangolin classification [4]. Up to January 2021, a total of 181 individuals were detected with the B.1.1.50 + P681H variant. Most of these individuals were randomly sampled from various districts across Israel, with no gender or age bias. The prevalence of the variant in the population decreased with time, from 12.7% out of 212 randomly sequenced sampled in November 2020, to 8.3% in December 2020 (out of 854 random samples), to 3.3% in January (out of

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2522 random samples) (Table 1). On the other hand, the B.1.1.7 variant, first identified in 2522 random samples) (Table 1). On the other hand, the B.1.1.7 variant, first identified in Israel in December 2020 and now the dominant lineage in Israel, increased with time, from Israel in December 2020 and now the dominant lineage in Israel, increased with time, from 0% in November to ~40% in January. 0% in November to ~40% in January. Table 1. Epidemiology of the B.1.1.50 + P681H variant. Frequency and patient-related information Vaccines 2021, 9, 616 Tableof the 1.B.1.1.50 Epidemiology + P681H variant.of the B.1.1.50 Ret. abroad + P681H is return variant. from Frequency abroad; Jud and & patient-related Sam is Judea and information Samaria. 5 of 9 of the B.1.1.50 + P681H variant. Ret. abroad is return from abroad; Jud & Sam is Judea and Samaria. November December January November December January Tablegender 1. Epidemiology of the B.1.1.50Male + P681H variant.10 Frequency and30 patient-related 25 information gender Male 10 30 25 of the B.1.1.50 + P681H variant.Female Ret. abroad is return from8 abroad; Jud24 & Sam is Judea23 and Samaria. Female 8 24 23 Unknown 9 17 35 Unknown November9 December17 35 January age 39.3 ± 21.3 33.8 ± 25.3 35.8 ± 22 age 39.3 ± 21.3 33.8 ± 25.3 35.8 ± 22 reasongender random Male 1027 55 30 80 25 reason randomFemale 27 8 55 24 80 23 outbreaksUnknown 9 10 17 35 outbreaks 10 ageret. abroad 39.3 ± 21.3 33.86± 25.3 35.83 ± 22 ret. abroad 6 3 districtreason Northern random 273 7 55 14 80 district Northern 3 7 14 outbreaksHaifa 4 2 10 5 ret.Haifa abroad 4 2 65 3 Central 4 19 32 districtCentral Northern 4 3 19 7 32 14 Tel Aviv 1 11 10 TelHaifa Aviv 1 4 11 2 10 5 JerusalemCentral 41 9 19 9 32 Jerusalem 1 9 9 JudTel & Aviv Sam 11 11 5 10 Jud & Sam 1 5 SouthernJerusalem 11 4 9 3 9 SouthernJud & Sam 1 4 3 5 Unknown 12 19 5 UnknownSouthern 12 1 19 4 5 3 Unknown 12 19 5 total B.1.1.50 + P681H 27 71 83 total B.1.1.50 + P681H 27 71 83 total B.1.1.50 + P681H 27 71 83 % of random 12.74 8.31 3.29 % of% random of random 12.74 12.74 8.31 8.31 3.29 3.29

total B.1.1.7 0 64 1008 totaltotal B.1.1.7 B.1.1.7 0 64 64 1008 1008

% of% random of random 0.00 7.49 7.49 39.97 39.97 % of random 0.00 7.49 39.97 3.2. Identification3.2. Identification of the ofP681H the P681H Mutation Mutation in Sewage in Sewage 3.2. Identification of the P681H Mutation in Sewage The P681HThe P681H mutation mutation was also was frequently also frequently identified identified by SARS-CoV-2 by SARS-CoV-2 whole whole genome genome The P681H mutation was also frequently identified by SARS-CoV-2 whole genome sequencingsequencing of samples of samples from fromwaste-water waste-water treatm treatmentent plants plants in nine in ninelocations locations across across Israel, Israel, sequencing of samples from waste-water treatment plants in nine locations across Israel, whichwhich were werecollected collected once oncea month a month from fromAugust August 2020 2020to January to January 2021 2021(Figure (Figure 1). As1). the As the which were collected once a month from August 2020 to January 2021 (Figure 1). As the P681HP681H mutation mutation is also is found also found in the in B.1.1.7 the B.1.1.7 variant, variant, its frequency its frequency has increased has increased in some in some P681H mutation is also found in the B.1.1.7 variant, its frequency has increased in some locationslocations since sincelate December late December 2020, 2020,following following the first the introduction first introduction of B.1.1.7 of B.1.1.7 into Israel. into Israel. locations since late December 2020, following the first introduction of B.1.1.7 into Israel. Notably,Notably, the mutation the mutation was already was already observed observed with witha 5% afrequency 5% frequency as early as early as October as October 2020 2020 Notably, the mutation was already observed with a 5% frequency as early as October 2020 in Rahatin Rahat (city (cityin the in South the South of Israel) of Israel) and in and November, in November, at a frequency at a frequency of 98% of 98%in Natanya in Natanya in Rahat (city in the South of Israel) and in November, at a frequency of 98% in Natanya and Haifa,and Haifa, located located at the at center the center and north and north of Israel, of Israel, respectively respectively (Figure (Figure 2). 2). and Haifa, located at the center and north of Israel, respectively (Figure 2). 3.3. Effective Neutralization of the B.1.1.50 + P681H Variant The neutralization potency of antibodies against the B.1.1.50 + P681H variant was compared to the neutralization of other strains commonly circulating in Israel. VERO-E6 cells were infected with (a) the B.1.1.50 + P681H variant (isolate hCoV-19/Israel/CVL-45176- P681H-ngs/2020), (b) an Israel WT strain (isolate hCoV-19/Israel/CVL-45526-ngs/2020), and (c) the B.1.1.7 variant (isolate hCoV-19/Israel/CVL-46879-ngs/2020). The different strains were pre-incubated with serial dilutions of serum samples obtained from individuals fully vaccinated with two doses of the Pfizer vaccine. The results demonstrate comparable neutralizations of the sera against the B.1.1.50 + P681H variant, the B.1.1.7 variant, and an Israel WT strain (Figure3). Moreover, negative sera from 10 individuals drawn prior to the pandemic showed no cross-reactivity with any of the virus isolates.

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FigureFigure 2. Identification Identiﬁcation of of the the P681H P681H mutation mutation in insewers sewers across across Israel. Israel. FrequencyFrequency of P681H of P681H mutation mutation in SARS-CoV-2 in SARS- CoV-2genomes genomes sequenced sequenced from nine from sewage nine samples sewage of samples wastewater of wastewater treatment plants treatment across plants Israel across each month Israel in each August month 2020– in AugustJanuary 2020–January 2021. The frequency 2021. The of frequencythe P681H ofmutation the P681H in each mutation region in was each estimated region was by estimatedmeasuring by the measuring fraction of the the fraction mutation from the total number of nucleotides mapped at this position (i.e., depth of sequencing). of the mutation from the total number of nucleotides mapped at this position (i.e., depth of sequencing). 3.3. Effective Neutralization of the B.1.1.50 + P681H Variant The neutralization potency of antibodies against the B.1.1.50 + P681H variant was compared to the neutralization of other strains commonly circulating in Israel. VERO-E6 cells were infected with (a) the B.1.1.50 + P681H variant (isolate hCoV-19/Israel/CVL- 45176-P681H-ngs/2020), (b) an Israel WT strain (isolate hCoV-19/Israel/CVL-45526- ngs/2020), and (c) the B.1.1.7 variant (isolate hCoV-19/Israel/CVL-46879-ngs/2020). The

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different strains were pre-incubated with serial dilutions of serum samples obtained from individuals fully vaccinated with two doses of the Pfizer vaccine. The results demonstrate comparable neutralizations of the sera against the B.1.1.50 + P681H variant, the B.1.1.7 variant, and an Israel WT strain (Figure 3). Moreover, negative sera from 10 individuals Vaccines 2021, 9, 616 7 of 9 drawn prior to the pandemic showed no cross-reactivity with any of the virus isolates.

Figure 3. Neutralization of thethe B.1.1.50B.1.1.50 ++ P681HP681H variant.variant. NeutralizationNeutralization assays were carriedcarried outout with VERO-E6VERO-E6 cellscells infected infected with with the the B.1.1.50 B.1.1.50 + P681H + P681H variant, variant, B.1.1.7 B.1.1.7 variant variant and anand Israel an Israel WT strain WT fromstrain Israel, from usingIsrael, sera using from sera fully from vaccinated fully vaccinate individuals.d individuals. On day 6,On plates day were6, plates colorized were overnightcolorized overnight with Gentian violet +4% formaldehyde solution for virus neutralization. Titers were cal- with Gentian violet +4% formaldehyde solution for virus neutralization. Titers were calculated by culated by qualitative measurements of the cytopathic effect for each patient. Bars represent the qualitative measurements of the cytopathic effect for each patient. Bars represent the geometric mean geometric mean titer (GMT) and 95% confidence intervals. titer (GMT) and 95% conﬁdence intervals.

4. Discussion and Conclusions The P681H mutation was observed as early as March 2020 in samples worldwide and also characterizes the globally spreading B.1.1.7 variant. Herein, we report a novelnovel locallocal variant withwith thethe P681HP681H mutation mutation that that emerged emerged from from the the locally locally circulating circulating B.1.1.50 B.1.1.50 lineage. line- Thisage. This local local B.1.1.50 B.1.1.50 + P681H + P681H variant variant was identifiedwas identified in 181 in sequenced 181 sequenced samples samples collected collected from Novemberfrom November 2020 2020 to January to January 2021 2021 in Israel. in Israel. Although Although first first detected detected in clinicalin clinical samples samples in Novemberin November 2020, 2020, the the P681H P681H mutation mutation had had already already been been identified identified in Israel in Israel in October in October 2020 via2020 sequencing via sequencing of sewage of sewage samples. samples. The 681 site is located in the vicinity of thethe furinfurin cleavagecleavage sitesite inin thethe SS protein.protein. It is postulated to enhance transmissibility of the virus by facilitating a conformational change in the the S S protein protein following following protease protease activity activity at atthe the cell cell membrane membrane [15]. [ 15Previous]. Previous serum serum neu- neutralizationtralization assays assays for forVSV-based VSV-based SARS-CoV-2 SARS-CoV-2 pseudoviruses pseudoviruses expressing expressing the the S protein with the P681H mutation demonstrated no significantsignificant changes in the titer required forfor neutralization compared to the unmutatedunmutated SS proteinprotein [[28].27]. Additionally, nono significantsignificant changes in the titer were needed for neutralizationneutralization of the WT SARS-CoV-2 compared to a viral samplesample fromfrom the the B.1.1.7 B.1.1.7 variant variant (in (in which which P681H P681H is one is ofone its of defining its defining mutations), mutations), using serausing from sera convalescent from convalescent patients orpatients individuals or individuals vaccinated vaccinated with the Moderna with the mRNA-1273 Moderna vaccinemRNA-1273 from vaccine Moderna from [28 ].Moderna Similar to[28]. these Similar reports, to these this studyreports, demonstrates this study demonstrates comparable neutralizationcomparable neutralization of the B.1.1.50 of + the P681H B.1.1.50 variant, + P681H an Israel variant, WT strainan Israel and WT the B.1.1.7strain and variant, the byB.1.1.7 sera variant, from vaccinated by sera from individuals. vaccinated individuals. The prevalence prevalence of of the the B.1.1.50 B.1.1.50 + +P681H P681H vari variantant declined declined with with time, time, from from ~12% ~12% in No- in Novembervember 2020 2020 to to3.2% 3.2% in inJanuary January 2021 2021 and and 0.9% 0.9% in in February February 2021 2021 (out (out of of 2843 randomly sequenced samples, data not shown). The dec declineline of this variantvariant alongalong withwith additionaladditional local lineages, such as B.1.1.50 and B.1.362, is attributedattributed to thethe increaseincrease inin prevalenceprevalence ofof the B.1.1.7 variant, which was first detected in Israel in December 2020 and is now the dominant variant in Israel, as in many other countries worldwide. Overall, as the B.1.1.50 + P681H variant was efficiently neutralized by vaccine-derived antibodies, similarly to local unmutated circulating strains, it was not associated with esca- lated infection or spread, and in light of the P681H mutation being observed in additional SARS-CoV-2 strains worldwide not defined as VOCs, this emerging local variant is cur- Vaccines 2021, 9, 616 8 of 9

rently not deﬁned as a VOC. Nevertheless, it is still monitored by routine next generation sequencing in Israel.

Author Contributions: Conceptualization, N.S.Z. and S.F.; methodology, N.S.Z., E.B., I.B.-O., V.I., M.W., Y.L. and M.M.; software, D.B.-I.; formal analysis, N.S.Z.; investigation, N.S.Z. and O.M.; resources, N.S.Z., M.M.,Y.L., E.M.; data curation, O.M.; writing—original draft preparation, N.S.Z.; writing—review and editing, N.S.Z., M.L., E.M., M.M., O.M.; visualization, N.S.Z.; supervision, N.S.Z., O.M.; project administration, N.S.Z., O.M., N.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of Chaim Sheba Medical Center (protocol code 7045-20-SMC, 24 March 2020). Informed Consent Statement: Not applicable. Data Availability Statement: All sequence was deposited to GISAID. Acknowledgments: The National Covid-19 Information & Knowledge Center. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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