key: cord-0706731-4p3robke authors: Sartor, Ivaine Tais Sauthier; Varela, Fernanda Hammes; Meireles, Mariana Rost; Kern, Luciane Beatriz; Azevedo, Thaís Raupp; Giannini, Gabriela Luchiari Tumioto; da Silva, Mariana Soares; Demoliner, Meriane; Gularte, Juliana Schons; de Almeida, Paula Rodrigues; Fleck, Juliane Deise; Zavaglia, Gabriela Oliveira; Fernandes, Ingrid Rodrigues; de David, Caroline Nespolo; Santos, Amanda Paz; de Almeida, Walquiria Aparecida Ferreira; Porto, Victor Bertollo Gomes; Scotta, Marcelo Comerlato; Vieira, Gustavo Fioravanti; Spilki, Fernando Rosado; Stein, Renato T.; Polese-Bonatto, Márcia title: Y380Q novel mutation in receptor-binding domain of SARS-CoV-2 spike protein together with C379W interfere in the neutralizing antibodies interaction date: 2022-01-16 journal: Diagn Microbiol Infect Dis DOI: 10.1016/j.diagmicrobio.2022.115636 sha: 45d33d9e97c3be8a117e7015859b0daac290ad64 doc_id: 706731 cord_uid: 4p3robke We aimed to describe the SARS-CoV-2 lineages circulating early pandemic among samples with S gene dropout and characterize the receptor-binding domain (RBD) of viral spike protein. Adults and children older than 2 months with signs and symptoms of COVID-19 were prospectively enrolled from May to October in Porto Alegre, Brazil. All participants performed RT-PCR assay, and samples with S gene dropout and cycle threshold < 30 were submitted to high-throughput sequencing (HTS). 484 out of 1,557 participants tested positive for SARS-CoV-2. The S gene dropout was detected in 7.4% (36/484) and a peak was observed in August. The B.1.1.28, B.1.91 and B.1.1.33 lineages were circulating in early pandemic. The RBD novel mutation (Y380Q) was found in one sample occurring simultaneously with C379W and V395A, and the B.1.91 lineage in the spike protein. The Y380Q and C379W may interfere with the binding of neutralizing antibodies (CR3022, EY6A, H014, S304). SARS-CoV-2 is a single RNA-stranded virus with high mutation rates. Strategies to mitigate the pandemic include the knowledge of its viral genome and expected mutations. These features could impact disease severity, virus transmission, and vaccine strategies [1, 2] . As the COVID-19 pandemic evolves, there has been concern about the emergence of new SARS-CoV-2 mutations in the receptor binding domain (RBD) from the S region, due to probable effects on both virus transmissibility and the generation of escape mutants from antibodies previously formed to heterologous lineages and vaccines [3] . Genetic alterations in the RBD of SARS-CoV-2 may improve the affinity of the virus to binding host cells, possibly increasing transmission rates [4, 5] and making this region a key target for potential therapies and diagnosis [6] . COVID-19 molecular diagnostic tests directed to the S gene use it as one of the RT-PCR multiple target-regions. Our aim was to measure the prevalence of the S dropout and characterize the SARS-CoV-2 mutations in the RBD region in a cohort during the early pandemic. A prospective cohort study enrolled adults and children seeking care at emergency rooms, outpatient clinics, or hospitalized in general wards or intensive care units (ICU) at Hospital Moinhos de Vento and Hospital Restinga e Extremo Sul, in Porto Alegre, Brazil. From May to early October 2020 were included participants presenting signs or symptoms suggestive of COVID-19 (cough, fever, or sore throat). The key exclusion criteria was a negative SARS-CoV-2 RT-PCR result or failure to sample collection. The study was performed in accordance with the Decree 466/12 of the National Health Council [7] and Clinical Practice Guidelines, after approval by the Hospital Moinhos de Vento IRB nº 4.637.933. All participants included in this study provided written informed consent. All participants performed qualitative RT-PCR assay (TaqManTM 2019-nCoV Kit v1, catalog number A47532, ThermoFisher Scientific, Pleasanton, California, EUA) to SARS-CoV-2 detection as described elsewhere [8] . Additionally, S gene dropout samples with cycle threshold less than 30 (Ct < 30.0) were submitted to high-throughput sequencing (HTS) using the Illumina MiSeq. RNA was extracted from naso-oropharyngeal swab samples and the reverse transcription reaction was performed using SuperScript IV reverse transcriptase kit (Thermo Fisher Scientific, Waltham, MA, USA). Libraries were prepared using QIAseq SARS-CoV-2 Primer Panel and QIAseq FX DNA Library UDI kit, according to the manufacturer instructions (Qiagen, Hilden, Germany). The QIAseq SARS-CoV-2 Primer Panel contains a PCR primer set for whole genome amplification of SARS-CoV-2 whose primer sequences were based on the ARTIC network nCov-2019. A pool of all of the normalized libraries was prepared and diluted to a final concentration of 8pM and sequenced on the Illumina MiSeq platform using the MiSeq Reagent kit v3 600 cycles (Illumina). FASTQ reads were imported to Geneious Prime, trimmed (BBDuk 37.25), and mapped against the reference sequence hCoV-19/Wuhan/WIV04/2019 (EPI_ISL_402124) available in EpiCoV database from GISAID [9]. Complete genome alignment was performed with the sequences generated. 59 Brazilian SARS-CoV-2 complete genomes and the reference sequence (EPI_ISL_402124) (>29 kb) were retrieved from the GISAID database using Clustal Omega. Maximum Likelihood phylogenetic analysis was applied under the General Time Reversible model allowing for a proportion of invariable sites and substitution rates in Mega X applying Wild type (Y380) and mutated spike protein sequences (Q380) were submitted to Bepipred 1.0 and 2.0 to detect putative humoral epitopes through HMMs and Random forest algorithms [10, 11] . To increase sensitivity we set a threshold of -0.2 (Bepipred 1.0) and 0.45 (Bepipred 2.0). The search in Immune Epitope Database (IEDB) considered T-cells epitopes for SARS-CoV-2 spike protein (region of 10 residues flanking the Y380Q) with 70% similarity in BLAST. The wild type SARS-CoV-2 surface glycoprotein sequence (NCBI accession number: YP_009724390) was submitted to BLAST and SwissModel tools. Template crystal candidates were evaluated by the GMQE, QMEAN, Z-score, and residues distribution in the Ramachandran plot, using ERRAT, PROCHECK, PDBsum, ModFold, SwissModel [14] [15] [16] [17] . Protein Data Bank (PDB) 7CWL (3.8Å) was chosen for the approach. Phyre-2 software was employed to homology modeling using the expert mode (one-to-one threading job) for constructing models based on wild protein (P0DTC2) and mutated sequences [18] . Electrostatic potential (EP) was verified through Delphi web server calculations and PIPSA [19, 20] . Residue exposure characteristics (hydrophobicity and the Accessible Solvent Surface Area -aSAS) were estimated using the Chimera interface [21] . Crystal complexes of the RBD region with antibodies were recovered from PDB. The LigPlot program was applied to infer protein-antibody interaction sites [22] . Hydrogen-bonds (Hbonds) inferences among the RBD domain and antibodies were calculated in the Chimera interface. Data normality assumptions were verified for continuous variables, and median values and interquartile ranges (IQR) were calculated. Pearson's Chi-square test was used to evaluate proportions between the identified and undetermined results from S gene target, on the epidemiological week; Fisher's exact test was used to compare the frequencies of S dropout considering outpatient and inpatient populations. All analyses were performed in R 3.5.0 statistical software. A total of 1,557 participants were screened and 484 were detected positive for SARS-CoV-2 (Supplementary Figure 1 ). Of these, 98 (20.2%) subjects were hospitalized and 386 (79.8%) were seen as outpatients only. S dropout was characterized as undetermined RT-PCR values for the S gene target, and detected values for ORF1ab and N target probes. We observed a total S dropout of 36/484 (7.4%), while ORF1ab and N gene targets showed no dropout ( Figure 1) . Additionally, an increase in frequency of undetermined results of the S gene target was identified on 10 th Wild C379 and Y380 residues of the RBD region were predicted as part of B-cell epitopes in the Bepipred 1.0 and 2.0, even after the amino acid substitutions. NetMHCpan4.1 returns seven binding sequences involving these sites, and two of them were also described as epitopes in the IEDB positive T-cell assays: SASFSTFKCY (for HLA-A*01:01 allele) and KCYGVSPTK (for HLA-A*03:01 allele). The wild sequence (SASFSTFKCY), predicted as a weak binder, turns to a non-binder when mutated (SASFSTFKWQ). The wild strong binder sequence (KCYGVSPTK) turns into a weak binder (KWQGVSPTK). Location of identified mutations in the spike protein is depicted in Figure 3A . Structural analysis revealed EP modifications (orange rectangle, Figure 3B ) on the LMM52630 models Figure 3B ). These results can be even more evident examining the surface distribution charges: the D614 and D839 wild residues are negatively charged (red pattern, Figure 3B ) and this pattern is disrupted in G614 and Y839 mutated residues; while the surface EP distribution and models conformation show more discreet modifications for the RBD region variants. mutation changed the direction from highly hydrophobic (2.5) to hydrophilic (-0.9). Altered residues in the RBD region, especially the C379W and Y380Q mutations, are located close to each other and likely gain strength, thus providing an overall shift to hydrophilic profile. The changing potential of these two close mutations induced to the buried A395 a more hydrophilic profile when compared to the ancestor (from 4.2 to 1.8). A structural investigation of more than 20 crystals of viral spike protein from PDB, revealed that the mutations C379W, Y380Q and V395A are in a contact area complexed with antibodies in the RBD region. And four crystals that presented Fab antibodies (fragment antigen-binding) are in contact with 379 and 380 RBD residues. Figure 4B It is well known that modifications in the EP and hydrophobicity distribution may interfere in protein-protein interaction. Interface regions are usually composed of residues presenting opposite charges and hydrophobic pairs, where small changes on these properties, in important functional sites, may impact canonical interactions [25] . NAbs are fundamental elements of the immune system against viral infections [26] , and the H-bonds of wild C379 and Y380 residues with the S304, CR3022, S2H97 and EY6A NAbs reinforce the importance of these regions. Therefore, the H-bond disruptions observed in W379 and Q380 substitutions plus the alteration in hydrophobicity disfavor the RBD-antibodies interactions. As the C379 residue is part of one (1/4) disulfide bonds in the RBD region (C379-C432) its disruption could generate instability since it contributes to β sheet conformation maintenance [27] . A previous study reported that the C379 and Y380 residues are part of an epitope for H014 antibody, which could sterically compete with the ACE2 host molecule for the RBD interaction [28] . It also reported an overlap among the binding epitopes for the H014 and CR3022 antibodies. The CR3022 monoclonal antibody neutralizes the RBD region of SARS-CoV-2, disrupts the prefusion spike conformation, and also competes sterically with ACE2 [29] without physically blocking it [30] . The recently described S2H97 is a potential NAb [31] that exhibits a notable tight binding even with divergent RBD regions from other Sarbecoviruses. Other SARS-CoV-2 NAbs that bind to the RBD were also described as nonoverlapping with the ACE2 binding site [32] . This study has some limitations. The small sample size in which HTS was feasible may limit any conclusion about the clinical severity related to the mutations found. Moreover, individuals were enrolled in a single city in Southern Brazil, which may limit the generality of our findings. Nonetheless, despite such limitations, a novel mutation (Y380Q) in the RBD region of SARS-CoV-2 spike protein was described. Analysis based on crystal structures reinforces the importance of the Y380 and C379 residues in the NAbs binding, and thus mutations in these regions may affect the interaction effectiveness between the NAbs and SARS-CoV-2 protein, as inferred by computational analysis. Our findings indicate that SARS-CoV-2 variants were circulating quite early in the community. A possible role of the new described mutation with clinical severity can be speculated, but further studies are needed to confirm this hypothesis. Studies assessing mutations and their relation to prognosis are necessary, and also to evaluate vaccine effectiveness in a challenging scenery that is continuously changing. Ivaine Tais Sauthier Sartor: Conceptualization, Investigation, Formal analysis, Data Curation, Visualization, Writing -Original Draft Conceptualization, Investigation, Visualization, Writing -Original Draft Formal analysis, Visualization, Writing -Original Draft Luciane Beatriz Kern: Resources, Validation, Investigation, Writing -Original Draft Thaís Raupp Azevedo: Resources, Validation, Investigation, Writing -Original Draft Resources, Validation, Investigation, Writing -Original Draft Validation, Investigation, Data Curation, Formal analysis, Visualization, Writing -Original Draft Meriane Demoliner: Validation, Investigation, Writing -Original Draft Juliana Schons Gularte: Validation, Investigation, Writing -Original Draft Validation, Investigation, Writing -Original Draft Juliane Deise Fleck: Validation, Investigation, Writing -Original Draft Resources, Investigation, Writing -Original Draft Ingrid Rodrigues Fernandes: Resources, Investigation, Writing -Original Draft Resources, Investigation, Writing -Original Draft Amanda Paz Santos: Resources, Investigation, Writing -Original Draft Conceptualization, Writing -Review & Editing Victor Bertollo Gomes Porto: Conceptualization, Writing -Review & Editing Marcelo Comerlato Scotta: Conceptualization, Visualization, Writing -Review & Editing, Supervision Conceptualization, Investigation, Writing -Review & Editing, Supervision Fernando Rosado Spilki: Conceptualization, Investigation, Writing -Review & Editing, Supervision Renato T. 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