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Mutations Associated withSARS-CoV-2 Variants of Concern,Benin, Early 2021Anna-Lena Sander,1 Anges Yadouleton,1 Edmilson F. de Oliveira Filho, Carine Tchibozo,Gildas Hounkanrin, Yvette Badou, Praise Adewumi, Keke K. René, Dossou Ange,Salifou Sourakatou, Eclou Sedjro, Melchior A. Joël Aïssi, Hinson Fidelia,Mamoudou Harouna Djingarey, Michael Nagel, Wendy Karen Jo, Andres Moreira-Soto,Christian Drosten, Olfert Landt, Victor Max Corman, Benjamin Hounkpatin, Jan Felix DrexlerIntense transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Africa mightpromote emergence of variants. We describe 10 SARSCoV-2 lineages in Benin during early 2021 that harboredmutations associated with variants of concern. Benin-derived SARS-CoV-2 strains were more efficiently neutralized by antibodies derived from vaccinees than patients,warranting accelerated vaccination in Africa.Genomic surveillance is key to elucidate coronavirus disease (COVID-19) transmission chainsand to monitor emerging severe acute respiratorysyndrome coronavirus 2 (SARS-CoV-2) variants asAuthor affiliations: Charité-Universitätsmedizin Berlin, corporatemember of Freie Universität Berlin, Humboldt-Universität zuBerlin, Institute of Virology, Berlin, Germany (A.-L. Sander,E.F. de Oliveira Filho, W.K. Jo, A. Moreira-Soto, C. Drosten,V.M. Corman, J.F. Drexler); Ecole Normale Supérieure deNatitingou, Natitingou, Benin (A. Yadouleton); UniversitéNationale des Sciences, Technologies, Ingénierie etMathématiques (UNSTIM), Cotonou, Benin (A. Yadouleton);Laboratoire des Fièvres Hémorragiques Virales du Benin,Cotonou (A. Yadouleton, C. Tchibozo, G. Hounkanrin, Y. Badou,P. Adewumi); Ministry of Health, Cotonou (K.K. René, D. Ange,S. Sourakatou, B. Hounkpatin); Conseil National de Lutte contre leVIH-Sida, la Tuberculose, le Paludisme, les IST et les Epidémies,Cotonou (E. Sedjro, M.A. Joël Aïssi, H. Fidelia); World HealthOrganization Regional Office for Africa, Health EmergenciesProgramme, Brazzaville, Democratic Republic of the Congo(M.H. Djingarey); Deutsche Gesellschaft für InternationaleZusammenarbeit, Bonn, Germany (M. Nagel); German Centrefor Infection Research (DZIF), associated partner CharitéUniversitätsmedizin Berlin, Berlin (C. Drosten, V.M. Corman,J.F. Drexler); TIB Molbiol Syntheselabor GmbH, Berlin (O. Landt)DOI: https://doi.org/10.3201/eid2711.211353sociated with partial or complete immune escape (1).Intense transmission likely promotes the emergenceof variants, including mutations in the gene encodingthe spike (S) protein, which is a major component ofall available COVID-19 vaccines (2). Genomic surveillance is notoriously weak in sub-Saharan Africa (Appendix Figure, panel A, p1.pdf). A total of 55SARS-CoV-2 lineages were described in West Africaas of May 25, 2021, considerably fewer than the 350lineages in affluent regions (Appendix Figure, panelB). We previously described 2 diverse lineages (A.4and B.1) in Benin early in the pandemic (3). In thisstudy, we analyzed SARS-CoV-2 genomic diversityin Benin 1 year later and assessed the ability of vaccinee-derived and patient-derived serum samples toneutralize SARS-CoV-2 variants.The StudyWe used 378 SARS-CoV-2–positive diagnostic respiratory samples tested at the reference laboratory inBenin during January 30–April 2, 2021, for genomicsurveillance. All samples with cycle threshold 36(Sarbeco E-gene assay; TIB Molbiol, https://www.tib-molbiol.de) were used for this study. To enablerapid prescreening of mutations known to affect theviral phenotype, we used 4 reverse transcription PCR(RT-PCR)–based single-nucleotide polymorphism(SNP) assays (VirSNiP; TIB Molbiol) targeting 9 hallmark mutations in 7 S codons of variants of concern(VOCs): B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma),and B.1.617.2 (Delta) (Table 1). A total of 374 (98.9%)samples selected for the study tested positive for 1mutation. Of those, 67.5% (255/378) showed the1These first authors contributed equally to this article.Emerging Infectious Diseases www.cdc.gov/eid Vol. 27, No. 11, November 20212899

DISPATCHESTable 1. Screened mutations, potential effects, and occurrence in severe acute respiratory syndrome coronavirus 2 variants,Benin, 2021SARS-CoV-2 variantSNPSpike ential effectsAlpha† B.1.525Beta† Gamma† P.2 P.3Delta†1del HV69/70Immune escape and enhanced viralxxinfectivity (4)E484KAntibody resistance (4)xxxxxN501YIncreased transmission (4)xxxx2V1176FHigher mortality rates‡xx3L452RAntibody resistance (4)x4K417TNo dataxK417NImmune escape (5)xP681HNo dataxP681RNo datax*SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SNP single-nucleotide polymorphism.†Variants of concern according to the World Health Organization.‡G. Hahn et al., unpub. data, .386714v2.69/70 deletion, 58.9% (223/378) the E484K mutation,33.9% (128/378) the N501Y mutation, 30.4% (115/378)the P681H mutation, 14.8% (56/378) the L452R mutation, and 0.3% (1/378) the K417N or P681R mutation.The K417T or V1176F mutations associated with theBeta and Gamma VOCs were not detected. Approximately 22.2% (84/378) of samples were typeable to1 of the lineages covered by the VirSNiP assays. According to SNP-based analyses, 14.8% (56/378) ofthe overall samples showed the mutation pattern ofthe Alpha variant, B.1.1.7, and 7.4% (28/378) of theB.1.525 variant. Frequent occurrence of the mutationsunder study suggests that earlier SARS-CoV-2 lineages not carrying those mutations have been replacedin Benin.Definite lineage designation relies on the fullgenome sequence. We selected 68 (9 typeable and59 nontypeable) samples according to unique mutational patterns covering the complete period of thestudy for a NimaGen/Illumina-based whole-genomesequencing workflow (Appendix Table 1). All near-full genomes generated within this study were deposited into GISAID (https://www.gisaid.org; accessionnos. EPI ISL 2932532–84 and EPI ISL 2958658–72).Lineage assignment using the Pangolin COVID-19Lineage Assigner version 3.0.2 (https://pangolin.cog-uk.io) confirmed SNP-based lineage prediction inall 9 typeable samples selected for whole-genome sequencing (Appendix Table 2). Despite robust lineageprediction based on unambiguous SNP-based results,our data demonstrate the limited use of VirSNiP assays for strain designation; however, these assayscan detect relevant mutations of currently circulating variants. The 68 Benin-derived near-completegenomes were designated to 10 unique lineages, suggesting higher genetic diversity in Benin than 1 yearbefore (3). During early 2021, lineages B.1.1.7 (22%),A.27 (19.1%), B.1.525 (17.6%), and B.1.1.318 (16.2%)were most prominent in Benin (Appendix Table 3).Despite presence of the mutation P681R (associatedwith the Delta VOC) in 1 sequence, that strain wastyped as A.23.1, and no Delta variant was found.Figure 1. Genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages in Benin, 2021. A)Nonsynonymous mutations of Benin-derived SARS-CoV-2 sequences across the full genome. B) Spike mutations occurring in theSARS-CoV-2 lineages circulating in Benin. Hallmark mutations of variants of concern are shown in color. Other mutations occurring inthe Benin-derived sequences are depicted in gray and summarized as others. ORF, open reading frame; RBD, receptor-binding domain.2900Emerging Infectious Diseases www.cdc.gov/eid Vol. 27, No. 11, November 2021

SARS-CoV-2 Variants of Concern, Benin, Early 2021These data are consistent with recent online sequencereports from West Africa (A.E. Augustin, unpub.data, .21256282v1; E.A. Ozer et al., unpub. 04.09.21255206v3). A 100% consensus sequence ofall 68 Benin-derived sequences showed 229 nonsynonymous nucleotide substitutions across the wholegenome; 57 (24.9%) occurred in the S protein (Figure1, panel A). Of note, variants with mutations in the Sprotein might alter the transmissibility and antigenicity of the virus (4). Internationally recognized VOCsto date share 16 S mutations in unique ns). The Benin-derived SARS-CoV-2 strains shared 10 unique Smutations reported in VOCs, although most of thosestrains were not defined as any VOC other than Alpha(Figure 1, panel B), suggesting convergent evolutionof key mutations across different lineages (D.P. Martin et al., unpub. data, .21252268v3; S. Cherian,unpub. data, .440932v2). Putative higher fitness mediated by genomic change was consistent with moremutations in predominant lineages than in lineagesfound at lower frequencies (Figure 1, panel B).Because S mutations, individually or in combination, have been shown to afford viral escapeto antibody-mediated immune responses, the highprevalence of variants with large numbers of thesemutations circulating in Benin was cause for concern. To investigate whether and to what extentFigure 2. PRNT results of severeacute respiratory syndromecoronavirus 2 (SARS-CoV-2)variants from Benin, 2021. Graphscompare results of neutralizationtests for naturally infected persons(A) and persons who receivedthe Pfizer-BioNTech vaccine(BNT162b2; https://www.pfizer.com) (B) against the B.1.153lineage from January 2020(Munich/ChVir929/2020 strain;GISAID [http://www.gisaid.org]accession no. EPI ISL 406862;Pangolin version 2021–05–19), theBeta strain (Baden-Wuertemberg/ChVir22131/2021; accessionno. EPI ISL 862149; B.1.351;Pangolin version 2021–05–19)and the B.1.1.7, B.1.214.2, B.1,and A.27 lineages isolated frompatients from Benin. Lines denotethe mean PRNT50 endpointtiter. Statistical significance wasdetermined by the Dunn’s multiplecomparisons test. Nonsignificantvalues are not shown for clarity ofpresentation. PRNT50, 50% plaquereduction neutralization test.Emerging Infectious Diseases www.cdc.gov/eid Vol. 27, No. 11, November 20212901

DISPATCHESTable 2. Hallmark mutations and PRNT50 results of Benin-derived severe acute respiratory syndrome coronavirus 2 lineages, Benin,2021Sample 1.214.2MutationsQ52R, Del HV69/70, Del Del HV69/70, Del Y144, L18F, L452R, N501Y, Ins R214TDR, Q414K,Y144, E484K, D614G,F490S, N501Y, A570D, A653V, H655Y, D796Y,D614G, T716IQ677H, F888LD614G, P681H, T716I,G1219VS982A, D1118HPatient-derived samplesMean titer (95% CI)23 (–12.4 to 58.4)35.5 (–12 to 83)65.6 (–46.6 to 177.7) 148.9 (–86.59 to 384.3)No. (%) neutralized3/6 (50)5/6 (83.3)4/6 (66.7)6/6 (100)Titer difference†52.2 (1.5-fold)39.79.7–73.6‡Vaccinee-derived samplesMean titer (95% CI)180.5 (102.8–258.1)156.2 (33.6–278.7)293.7 (57.1–530.2)698.3 (446.8–949.9)No. (%) neutralized7/7 (100)7/7 (100)7/7 (100)7/7 (100)Titer difference†136.716123.5–381.1‡*PRNT50, 50% plaque reduction neutralization test.†Compared to variant B.1.153.‡Lower titers against the early isolate compared with this Benin-derived isolate.SARS-CoV-2 variants circulating in Benin and WestAfrica (5) evade neutralizing antibody responses, weisolated 4 lineages with unique mutational patterns(Table 2): an A.27 lineage isolate harboring the N501Ymutation; a B.1 isolate harboring the 69/70 deletionand the E484K and D614G mutations; a B.1.1.7 lineageisolate harboring the 69/70 deletion and the N501Y,D614G, and P681H mutations; and a B.1.214.2 lineageharboring the Q414K and D614G mutations (Figure2). Additional isolation attempts of strains belonging to the frequently detected B.1.525 and B.1.318lineages failed, likely because of degradation after repeated freeze-thaw cycles under tropical conditions.We tested neutralization potency of 6 serum samplesfrom patients in Benin taken 8 days after RT-PCR–confirmed SARS-CoV-2 infection during early 2020(6) and another 7 serum samples from persons inEurope 4 weeks after receiving the second dose ofthe Pfizer/BioNTech vaccine (BNT162b2; https://www.pfizer.com) (Appendix Table 4). Sampling wasapproved by the ethics committee of the Benin Ministry of Health (approval no. 030/MS/DC/SGM/DNSP/CJ/SA/027SGG2020) and of Charité-Universitätsmedizin Berlin (approval nos. EA1/068/20and EA4/245/20). We compared neutralization titers with a SARS-CoV-2 strain (B.1.153) from January2020 and the Beta strain (B.1.351) known to evade antibody-mediated neutralization (7). Despite the earlysampling time after RT-PCR confirmation of SARSCoV-2 infection, all 6 serum specimens from patientsin Benin efficiently neutralized the early SARS-CoV-2isolate carrying only the D614G mutation. In contrast,only 3 of those 6 serum specimens neutralized the B.1isolate, the only isolate with the E484K mutation (Figure 2, panel A). Among the serum specimens fromvaccinated persons, all neutralized the B.1 isolate,albeit at 1.5-fold lower titers than the early lineage2902B.1.153 isolate (by Friedman test and Dunn’s multiplecomparisons test; p 0.99) (Figure 2, panel B). Thosedata were consistent with a recent report describingefficient neutralization of a B.1.525 strain from Nigeria by vaccinee-derived serum specimens (8). Of note,another strain classified as B.1.214.2 was neutralizedmore efficiently than all other tested lineages (Figure2), highlighting that not every mutation in circulatinglineages affords reduced antibody-mediated neutralization. Other hypothetically present fitness advantages of such strains will require detailed virologicinvestigation.Our study is limited by patient-derived samplestaken an average of 8 days after infection (7), whichcould imply incomplete maturation of antibodies.However, similar neutralization patterns betweenpatient-derived and vaccinee-derived serum specimens suggest robustness of our data. Another limitation is that vaccinee-derived serum samples originated exclusively from Europe. Vaccine responses varybetween populations, possibly influenced by geneticbackground and immune-modulating diseases (e.g.,malaria or HIV) (9), highlighting the importance oftesting serum samples from vaccinees in Africa forfuture studies. Of note, the efficacy trial of the Pfizer/BioNTech vaccine enrolled 40,000 participants, only 800 of whom were from Africa, and all of those fromSouth Africa (10).ConclusionsOur data highlight the importance of ongoingmonitoring of population immunity to emergingSARS-CoV-2 variants in Africa and of using serumspecimens from local settings for phenotypic characterizations. Vaccination programs in Africa should beaccelerated urgently, emphasizing the importance ofglobal access to vaccines.Emerging Infectious Diseases www.cdc.gov/eid Vol. 27, No. 11, November 2021

SARS-CoV-2 Variants of Concern, Benin, Early 2021AcknowledgmentsWe thank Sebastian Brünink, Arne Kühne, Ben Wulf, andAntje Kamprad for support.This work was funded by the Deutsche Gesellschaft fürInternationale Zusammenarbeit (GIZ) GmbH (projectnumber 81263623). This study is also based on researchfunded in part by the Bill & Melinda Gates Foundation(grant ID INV-005971). The findings and conclusionscontained within are those of the authors and do notnecessarily reflect positions or policies of the Bill &Melinda Gates Foundation.O.L. is the owner of TIB Molbiol, the company developingand marketing SARS VirSNiP assays.2.3.4.5.6.About the AuthorMs. Sander is a PhD student at the Institute of Virologyat Charité-Universitätsmedizin, Berlin, Germany; hermain research interest is the evolution of newlyemerging viruses. Dr. Yadouleton is a medicalentomologist in the Centre de Recherche Entomologiquede Cotonou, Benin, head of the Laboratoire des FièvresHémorragiques in Cotonou, and a teacher at theUniversity of Natitingou, Benin; his research interestsinclude mosquito control and the diagnosis of viralhemorrhagic fevers.7.8.9.10.References1.Warmbrod KL, West R, Frieman M, George D, Martin E, Rivers C. Staying ahead of the variants: policyrecommendations to identify and manage current and futurevariants of concern. Baltimore (MD): Johns Hopkins Centerfor Health Security; 2021 Feb 16 [cited 2021 May rk/publications/staying-ahead-of-the-variantsJo WK, Drosten C, Drexler JF. The evolutionary dynamics ofendemic human coronaviruses. Virus Evol. 2021;7:veab020.Sander AL, Yadouleton A, Moreira-Soto A, Tchibozo C,Hounkanrin G, Badou Y, et al. An observational laboratorybased assessment of SARS-CoV-2 molecular diagnostics inBenin, Western Africa. MSphere. .00979-20Harvey WT, Carabelli AM, Jackson B, Gupta RK,Thomson EC, Harrison EM, et al.; COVID-19 Genomics UK(COG-UK) Consortium. SARS-CoV-2 variants, spikemutations and immune escape. Nat Rev Microbiol.2021;19:409–24. https://doi.org/10.1038/s41579-021-00573-0Zhou D, Dejnirattisai W, Supasa P, Liu C, Mentzer AJ,Ginn HM, et al. Evidence of escape of SARS-CoV-2variant B.1.351 from natural and vaccine-induced sera. Cell.2021;184:2348–2361.e6. https://doi.org/10.1016/j.cell.2021.02.037Sanyang B, Kanteh A, Usuf E, Nadjm B, Jarju S, Bah A, et al.COVID-19 reinfections in The Gambia by phylogeneticallydistinct SARS-CoV-2 variants-first two confirmed eventsin west Africa. Lancet Glob Health. (21)00213-8Yadouleton A, Sander AL, Moreira-Soto A, Tchibozo C,Hounkanrin G, Badou Y, et al. Limited specificity of serologictests for SARS-CoV-2 antibody detection, Benin, WesternAfrica. Emerg Infect Dis. 2021;27:2020. 10.3201/eid2701.203281 https://doi.org/10.3201/eid2701.203281Liu J, Liu Y, Xia H, Zou J, Weaver SC, Swanson KA, et al.BNT162b2-elicited neutralization of B.1.617 and otherSARS-CoV-2 variants. Nature. 2021. https://doi.org/10.1038/s41586-021-03693-yKollmann TR. Variation between populations in the innateimmune response to vaccine adjuvants. Front Immunol.2013;4:81. https://doi.org/10.3389/fimmu.2013.00081Polack FP, Thomas SJ, Kitchin N, Absalon J, Gurtman A,Lockhart S, et al.; C4591001 Clinical Trial Group. Safety andefficacy of the BNT162b2 mRNA Covid-19 vaccine. N Engl JMed. 2020;383:2603–15. https://doi.org/10.1056/NEJMoa2034577Address for correspondence: Jan Felix Drexler, Helmut-RuskaHaus, Institute of Virology, Campus Charité Mitte, Charitéplatz 1,10098 Berlin, Germany; email: [email protected] Infectious Diseases www.cdc.gov/eid Vol. 27, No. 11, November 20212903

Author affi liations: Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Institute of Virology, Berlin, Germany (A.-L. Sander, . DNSP/CJ/SA/027SGG2020) and of Charité-Uni-versitätsmedizin Berlin (approval nos. EA1/068/20 and EA4/245/20). We compared neutralization ti-