Emergence of Q493R mutation in SARS-CoV-2 spike protein during bamlanivimab/etesevimab treatment and resistance to viral clearance
Authors: Aurélie Guigon, Emmanuel Faure, Chloé Lemaire, Marie-Charlotte Chopin, Claire Tinez, Ady Assaf, Mouna Lazrek, Didier Hober, Laurence Bocket, Ilka Engelmann, Enagnon Kazali Alidjinou
Journal: Journal of Infection (2021)

Data from clinical trials suggest that monoclonal antibody (mAb) treatments can prevent deaths and severe disease among people with mild to moderate COVID-19. Recently, a cocktail of two mAbs- bamlanivimab and etesevimab- was reported to cut the risk of hospitalization and death by 87%.1Gottlieb Robert L. Ajay N. Peter C. Joseph B. Barry H. Jason M. et al.Effect of bamlanivimab as monotherapy or in combination with etesevimab on viral load in patients with mild to moderate COVID-19: a randomized clinical trial.JAMA. 2021; 325: 632-644https://doi.org/10.1001/jama.2021.0202Google Scholar The European Medicine Agency (EMA) recommends the use of this combination in patients who do not require supplemental oxygen, and who are at high risk of progression to severe disease.2EMA. Assessment report. Eli Lilly and Company Limited use of bamlanivimab and etesevimab for the treatment of COVID-19 2021 https://www.ema.europa.eu/en/documents/referral/eli-lilly-company-limited-antibody-combination-bamlanivimab/etesevimab-covid19-article-53-procedure-assessment-report_en.pdf. Accessed August 2nd, 2021.Google Scholar Since SARS-CoV-2 Spike protein is the target of antibody-based therapeutics, they all suffer from one major risk: mutational escape of the Spike protein.3Daniele F. Fabrizio M. Neutralising antibody escape of SARS-CoV-2 spike protein: risk assessment for antibody-based Covid-19 therapeutics and vaccines.Rev Med Virol. 2021; (rmv.2231)https://doi.org/10.1002/rmv.2231Google Scholar We here report a well-characterized case of the emergence of SARS-CoV-2 Spike escape. Q493R mutation during bamlanivimab/etesevimab treatment. A 63-year-old patient was diagnosed in July 2017 with cutaneous T-cell lymphoma (mycosis fungoides). The patient underwent several treatments including carmustine, methotrexate, bexarotene, and extracorporeal photopheresis. Tumoral extension to lymph nodes (stage III) led to a CHOEP chemotherapy regimen. An allogeneic hematopoietic stem cell transplantation was then performed in January 2021, complicated by the occurrence of a graft-versus-host disease. A COVID-19 screening was performed in April 2021, due to an intrafamilial exposure, and a positive RT-PCR result was found on a nasopharyngeal (NP) specimen. The patient developed mild symptoms such as runny nose and dry cough. A treatment with bamlanivimab (700 mg) /etesevimab (1400 mg) was initiated. Sequential NP samples were collected for treatment follow-up. SARS-CoV-2 RNA detection and E484K mutation screening were performed with the AllplexTM SARS-CoV-2 Variants I Assay (Seegene, Eurobio®). For the viral whole genome sequencing, total RNA extraction was performed using the MGIEasy Nucleic Acid Extraction Kit on the MGISP-960 instrument (BGI®). The libraries were prepared using Illumina® COVIDSeq protocol, and paired-end sequencing with 150 bp read length was carried out on NextSeq 550 platform. Data were processed using DRAGEN COVIDSeq Test Pipeline 1.0.0 (Illumina®). Clades and lineages were assigned to the genomes according to the Nextstrain nomenclature (version 1.0.0) and the PANGOLIN package (version 2021.06.15), respectively. Mutation analysis of the Spike region was provided by Nextstrain and GISAID CoVsuver tool. Serum samples were also collected for antibody testing. Qualitative detection of anti-S antibodies was done with the Wantai SARS-CoV-2 Ab ELISA kit (Eurobio®) and the Quantivac. ELISA Anti SARS-CoV-2 (Euroimmun®) was used for antibody quantification. Overall, the clinical evolution was good regarding COVID-19. The patient was discharged from hospital just after the initiation of bamlanivimab/etesevimab treatment which was well tolerated, and was followed as outpatient in the infectious disease department. She experienced prolonged respiratory symptoms with dyspnea and cough; however, no worsening of the status was observed and patient did not require oxygen therapy or hospitalization for COVID-19 symptoms. Furthermore, chest X-ray and biological inflammatory markers remained normal. Virological data are summarized in Table 1, and show that the viral clearance was not efficient, with a viral load which remains high almost 40 days after the initiation of treatment. Viral whole genome sequencing identified the widely circulating B.1.1.7 lineage (20I/501Y.V1) SARS-CoV-2. The spike mutation analysis did not detect the E484K mutation during the follow-up; however, an A → G mutation was observed at nucleotide position 23,040 from day 15 post-treatment leading to Q493R amino-acid substitution in the spike protein. Good quality sequences were obtained in all tested samples, with a genome coverage between (99.99 and 100%), and a median number of reads per nucleotidic position between 174 and 3465. In addition, a focus on the specific position showed a very good coverage between 142X and 2516X (See Table 2).Table 1Virological data during treatment follow-up.Virological dataBamlanivimab/Etesevimab treatmentDay post treatment−2 (diagnosis)0715212739CT value (RDRP gene, Allplex assay)18.69.920.615.515.114.324.8SARS-CoV-2 lineageNAB.1.1.7B.1.1.7B.1.1.7B.1.1.7NAB.1.1.7Presence of E484K/Q mutation (Yes/No)NoNoNoNoNoNoNoAmino-acid at Spike position 493NAQQRRNARNumber of reads at Spike nucleotide position 23,040 (total number of reads)NA177A (177)142A (142)169 G (169)2506 G (2516)NA935 G (936)GISAID reference for viral genome sequenceNAEPI_ISL_ 2,143,435EPI_ISL_ 2,227,230EPI_ISL_ 2,361,537EPI_ISL_ 2,444,359NAEPI_ISL_ 2,646,207Total antibody testing (qualitative, Wantai) (Index)NANA11.613NA10.312.5IgG quantification (Euroimmun) (binding activity units/mL)NANA>1920>1920NA>1920>1920 Open table in a new tab Table 2A→G mutation at nucleotidic position 23,040. Open table in a new tab Furthermore, antibody testing confirmed the presence of high levels of anti-Spike IgG antibodies. This observation is clearly compatible with a reduced efficacy or a lack of efficacy of the bamlanivimab/etesevimab treatment. In the BLAZE-1 trial (phase 2), a combination treatment with 2800 mg of bamlanivimab and 2800 mg of etesevimab led to viral load change of −4.37 and –17.91 from baseline to day 11 and day 29 post-treatment, respectively. A Ct value lower than 27.5 on day 7 post-treatment was considered as persistently high viral load.1Gottlieb Robert L. Ajay N. Peter C. Joseph B. Barry H. Jason M. et al.Effect of bamlanivimab as monotherapy or in combination with etesevimab on viral load in patients with mild to moderate COVID-19: a randomized clinical trial.JAMA. 2021; 325: 632-644https://doi.org/10.1001/jama.2021.0202Google Scholar The resistance to treatment observed is this case is probably associated with the Q439R substitution that emerged in the Spike protein. The high initial viral load and the viral on-treatment replication could have contributed to the occurrence of this mutation. Changes in the spike protein can significantly alter the efficacy of mAbs, and the most dangerous for immune escape are the ones occurring within the receptor binding domain (RBD).3Daniele F. Fabrizio M. Neutralising antibody escape of SARS-CoV-2 spike protein: risk assessment for antibody-based Covid-19 therapeutics and vaccines.Rev Med Virol. 2021; (rmv.2231)https://doi.org/10.1002/rmv.2231Google Scholar In-vitro studies have identified mainly six amino-acid substitutions corresponding to four positions (E484D/K/Q, F490S, Q493R, and S494P) that led to a reduced susceptibility to bamlanivimab, while six amino-acid substitutions at three positions (K417N, D420N, and N460K/S/T/Y) were shown to be critical for etesevimab. More interestingly, a retention of susceptibility to the other antibody alone was observed, with the exception of the Q493R substitution. A pseudotyped virus-like particle assay showed for variants harboring E484K, E484Q, and Q493R substitutions, a reduced susceptibility to bamlanivimab and etesevimab combination of 17-fold, 22-fold, and > 100-fold, respectively.2EMA. Assessment report. Eli Lilly and Company Limited use of bamlanivimab and etesevimab for the treatment of COVID-19 2021 https://www.ema.europa.eu/en/documents/referral/eli-lilly-company-limited-antibody-combination-bamlanivimab/etesevimab-covid19-article-53-procedure-assessment-report_en.pdf. Accessed August 2nd, 2021.Google Scholar,4FDA. Fact sheet for health care providers emergency use authorization (EUA) of Bamlanivimab and Etesevimab 2021 https://www.fda.gov/media/145802/download. Accessed August 2nd, 2021.Google Scholar The escape site Q493 seems thus to be the most critical for the bamlanivimab and etesevimab cocktail. It was recently reported that this site is not in the receptor-binding ridge, but in a region of joint structural overlap by both antibodies, such that a substitution leading to a positively charged residue (R, K) may directly affect binding by each antibody.5Starr Tyler N. Greaney Allison J. Dingens Adam S. Bloom Jesse D. Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016.Cell Rep Med. 2021; 2100255https://doi.org/10.1016/j.xcrm.2021.100255Google Scholar As we prepare this manuscript, another team also described a similar case using Sanger sequencing of the viral spike region, with a follow-up period of 2 weeks.6Daniele F. Federica N. Angelo G. Francesco D. Dalla G.D. Andreina B. et al.Emergence of SARS-COV-2 Spike Protein Escape Mutation Q493R after Treatment for COVID-19.Emerg Infect Dis. 2021; 27https://doi.org/10.3201/eid2710.211538Google Scholar In conclusion, the emergence of the spike protein Q493R mutation can lead to virological failure during bamlanivimab/etesevimab treatment, and should be rapidly screened in nonresponders, especially when immunocompromized. This report was approved by the Institutional data protection authority of CHU Lille. Informed consent was obtained from the patient and data have been anonymized as much as possible.