key: cord-0307922-fygojft9
authors: Rockett, R. J.; Basile, K.; Maddocks, S.; Fong, W.; Agius, J. E.; Johnson-Mackinnon, J.; Arnott, A.; Chandra, S.; Gall, M.; Draper, J. L.; Martinez, E.; Sim, E. M.; Lee, C.; Ngo, C.; Ramsperger, M.; Ginn, A. N.; Wang, Q.; Fennell, M.; Ko, D.; Lim, L.; Gilroy, N.; Sullivan, M. V.; Chen, S. C.-A.; Kok, J.; Dwyer, D. E.; Sintchenko, V. L.
title: RESISTANCE CONFERRING MUTATIONS IN SARS-CoV-2 DELTA FOLLOWING SOTROVIMAB INFUSION
date: 2021-12-21
journal: nan
DOI: 10.1101/2021.12.18.21267628
sha: 418117e401da3c955814b59db3d90d09d428c399
doc_id: 307922
cord_uid: fygojft9

Several Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) neutralising monoclonal antibodies (mAbs) have received emergency use authorisation by regulatory agencies for treatment and prevention of Coronavirus Disease 2019 (COVID-19), including in patients at risk for progression to severe disease. Here we report the persistence of viable SARS-CoV-2 in patients treated with sotrovimab and the rapid development of spike gene mutations that have been shown to confer high level resistance to sotrovimab in vitro. We highlight the need for SARS-CoV-2 genomic surveillance in at risk individuals to inform stewardship of mAbs use and prevent potential treatment failures.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) which causes Coronavirus disease 2019 has spread rapidly worldwide causing over 258 million infections and 5 million deaths 1 , prompting the development of vaccines and antiviral agents at an unprecedented pace. 2 Several SARS-CoV-2-neutralizing monoclonal antibodies (mAbs) have been developed and received emergency use authorisation by regulatory agencies with additional mAbs currently advancing through phase 3 clinical trials. These therapeutics have been registered for treatment of mild to moderate COVID-19 disease in those at risk of progressing to severe disease. These agents target the SARS-CoV-2 spike (S) glycoprotein which consists of two functional subunits: S1 which includes the receptor-binding domain (RBD) and N-terminal domain (NTD) and mediates host attachment, and S2 subunit responsible for fusion of the virus and cellular membranes. The emergence of SARS-CoV-2 variants of concern (VOC) carrying mutations and deletions in the RBD, NTD and S2 subunit has highlighted the need for a more targeted utilization of these advanced therapeutics. Several mAbs cocktails (e.g., bamlanivimab/etesevimab) appear to be have reduced activity against viruses with the E484K and K417N/T mutations found in the lineages B.1.525, B.1.526 and VOC. 3 Other mAbs, such as sotrovimab (VIR-7831, GlaxoSmithKline, Australia Pty Ltd), target more conserved viral epitopes.

Sotrovimab is a human engineered monoclonal antibody that neutralizes SARS-CoV-2 and other sarbecoviruses, including SARS-CoV-1. 4 Sotrovimab acts by binding to a conserved epitope within the RBD, resulting in virus neutralization. The conservation of the epitope targeted by sotrovimab is supported by preservation of its activity in vitro against SARS-CoV2 VOC Alpha, Beta and Delta; however sotrovimab effectiveness is yet to be determined for the Omicron VOC. 5 Sotrovimab's safety and efficacy was evaluated in the phase 3, multicenter, randomised, double-blind, placebo-controlled COMET-ICE (COVID-19 Monoclonal Antibody Efficacy Trial-Intent to Care Early) trial. Trial recruitment targeted high-risk adults with symptomatic COVID-19 and interim results demonstrated a reduction in the risk of hospitalization (for >24 hours) or death from 7% in the placebo group to 1% in the sotrovimab group (85% relative risk reduction). Whilst evidence of sotrovimab effectiveness to prevent severe COVID-19 led to its approval for emergency use in the US, Singapore, Europe and . 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 Canada, Australia was one of the first countries to issue formal regulatory approval. [6] [7] [8] However, the use of SARS-CoV-2-specific monoclonal antibodies targeting a single viral epitope warrants caution as in vitro [9] [10] [11] and clinical case studies have demonstrated rapid development of mutations conferring resistance after exposure to various SARS-CoV-2 mAbs. [12] [13] [14] [15] [16] [17] Indeed, the acquisition of mutations in the sotrovimab target epitope at S protein amino acid positions 335-361 was reported during COMET-ICE. 4 Phenotypic characterization of these mutations demonstrated that high-level sotrovimab resistance (100 to 295-fold reduction in neutralization) was conferred by E340K/A/V, while a 192 to 304-fold reduction was also noted with the appearance of mutation P337L/K/R. 12 The acquisition of consensus mutations at S:E340K/A was also documented for 4 of 45 participants within the COMET-ICE trial. Observations of induced in vitro and clinical resistance has prompted the development of mAbs cocktails that simultaneously target multiple SARS-CoV-2 epitopes. 13 Here we report the rapid development of mutations in vivo that have been shown to confer high level resistance to sotrovimab in vitro and present a template for genomics guided use of mAbs.

A total of 100 patients received sotrovimab between 22 nd August 2021 and 13 th November 2021 at a single center in Australia. Sotrovimab was provisionally registered for use by the Australian Therapeutics Goods Administration in August 2021, with limited supply.

Treatment, a single 500mg infusion, was targeted at patients within five days of symptom onset who presented with risk factors for progression to severe disease. 4 Of the 100 patients that received sotrovimab, 23 had persistent SARS-CoV-2 RNA detected by reverse transcriptase -real time polymerase chain reaction (RT-PCR) for more than 10 days post-infusion (Supplementary Figure S1 ). A total of 68 patients did not have a follow-up SARS-CoV-2 RT-PCR test following treatment. Longitudinally-collected respiratory tract specimens pre-and post-sotrovimab were available for eight patients (35%) of the persistently RT-PCR positive patients and were thus investigated in this study (R001-R008).

Case demographic and clinical (co-morbidities, treatment and COVID-19 vaccine status) information was compiled for the study cohort (Table 1, Supplementary Table S1 ). Complete COVID-19 vaccination was defined as two doses of BNT162b2 (Comirnaty, Pfizer/BioNTech) received at least 7 days before testing positive to SARS-CoV-2 and in accordance with COVID-19 local vaccination recommendations. 14 Partial vaccination was defined as either one dose of vaccine or receipt of the second dose <7 days from SARS-CoV-2 RNA detection by RT-PCR.

Cases R009 and R010 were identified as asymptomatic household contacts of study cohort case 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 December 21, 2021. ; https://doi.org/10.1101/2021.12.18.21267628 doi: medRxiv preprint R001 and were also included in the study cohort (Supplementary Table S1 ). Ethical and governance approval for the study was granted by the Western Sydney Local Health District Human Research Ethics Committee (2020/ETH02426) and (2020/ETH00786) SARS-CoV-2 culture. Respiratory tract specimens that had detectable SARS-CoV-2 RNA by RT-PCR were cultured in vero E6 cells expressing transmembrane serine protease 2 (VeroE6/TMPRSS2; JCRB1819) as previously outlined (Supplementary Figure S2) . 15 

Tiling PCR was used to amplify the entire SARS-CoV-2 genome from RNA extracts of clinical specimens using primers outlined in the Midnight sequencing protocol. 16 Each PCR included 12.5µL Q5 High Fidelity 2x Master Mix (New England Biolabs), 1.1µL of either pool 1 or pool 2 10µM primer master mix, 2.5µL of template RNA and molecular grade water was added to generate a total volume of 25µL. Cycling conditions were initial denaturation at 95°C for 2 min, then 35 cycles of: 95°C for 30s, 65°C for 2 min . 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

The copyright holder for this this version posted December 21, 2021. ; https://doi.org/10.1101/2021.12.18.21267628 doi: medRxiv preprint 45s, and a final extension step of 75°C for 10 min. Pool 1 and pool 2 amplicons were combined and purified with a 1:1 ratio of AMPureXP beads (Beckman Coulter) and eluted in 30µL of RNAase free water. Purified products were quantified using Qubit™ 1x dsDNA HS Assay Kit (Thermo Fisher Scientific) and diluted to the desired input concentration for library preparation. Sequencing libraries were prepared using Nextera XT (Illumina) according to the manufacturer's respective instructions and pooled with the aim of producing 1x10 6 reads per library. Sequencing libraries were then sequenced with paired end 76 bp chemistry on the iSeq or MiniSeq (Illumina) platforms.

Bioinformatic analysis. Raw sequence data were processed using an in-house quality control procedure prior to further analysis as described previously. 17, 18 De-multiplexed reads were quality trimmed using Trimmomatic v0.36 (sliding window of 4, minimum read quality score of 20, leading/trailing quality of 5 and minimum length of 36 after trimming). 19 Briefly, reads were mapped to the reference SARS-CoV-2 genome (NCBI GenBank accession MN908947. previously been highlighted as problematic were monitored. 22 To ensure the accuracy of variant calls only high-quality genomes with >90% genome coverage and a mean depth of >1000x were included. The MFV calls were excluded in the base pair either side of the 5' or 3'-end of indels due to potential mis-mapping. SARS-CoV-2 lineages were inferred using Phylogenetic Assignment of Named Global Outbreak LINeages v1.2.86 (PANGO and PLEARN). 23, 24 Representative SARS-CoV-2 genomes collected between July to November 2021 (n=1,300, ≥27,000bp in length) were downloaded from Global initiative on sharing all influenza data (GISAID) 25 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Figure S2) . Genomes defined by PANGO as the Delta lineage (n=205) were used to contextualise sotrovimab resistant specimens generated in this study (n=50) ( Figure 1B) . The GISAID and New South Wales (NSW) genomes were aligned with MAFFT v7.402 (FFT-NS-2, progressive method). 27 Phylogenetic analysis was performed using the maximum likelihood approach (IQTree v1.6.7 (substitution model: GTR+F+R2) with 1,000 bootstrap replicates. 28 Graphs were generated using RStudio (version 3.6.1) and phylogenetic trees were constructed using the R package ggtree. 29 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

A median viral load of 142,619 copies/µL of SARS-CoV-2 RNA extract (range 2-8.2x10 8 copies/µl) was detected in longitudinal study cohort specimens collected between seven days pre-and 37 days post-sotrovimab infusion.

Longitudinal fluctuations in the viral load were observed for all cohort members ( Figure 1B, Supplementary Data S1). No significant difference in SARS-CoV-2 viral load was detected when comparing the viral loads pre-and post-sotrovimab (p=0.07508) or between cases with and without resistant mutations after sotrovimab treatment (p=0.01046) ( Figure 1C , Supplementary Data S1). A substantial drop in viral load was noted in two cases (R004 & R002) that subsequently rebounded after resistance mutations were detected.

Consensus genomes were recovered for 50/59 clinical specimens with median genome coverage and depth of 98.2% and 5,035.5x respectively (sequencing coverage min 92.0% max 99.9%, depth min 1,784.2 max 6,573.0) (Supplementary Data S1). All genomes were found to belong to Pangolin lineage AY.39.1, a sub-lineage of the Delta VOC that currently predominates in Australia and globally ( Figure 1A) . Specimens from which high-quality SARS-CoV-2 genomes were unable to be obtained had significantly lower viral loads (cohort median Ct 24.79 compared to Ct 34.17 for failed genomic specimens p=<0.0000.1) (Figure 1 .

Supplementary Data S1). Four of the eight patients in this study acquired previously defined RBD mutations between 6-13 days after sotrovimab treatment (Table 1; Figure 1C ). All but one case developed the S:E340K mutation which has previously demonstrated the highest resistance to sotrovimab. Read frequencies of S:E340K/A/V mutations generally increased over the course of infection, in two cases (R002 & R003) the proportion of the viral population carrying these mutations exceeded 75% at days 7 and 13, respectively, and remained fixed at subsequent time-points. In cases R002 and R004, S:E340K was initially detected in increasing frequency, subsequently interchanging between S:E340A and S:E340V. In addition, R002 developed a MFV at P337L after fixation of the S:E340K mutation 24 days after sotrovimab.

R004 died 37 days after sotrovimab treatment due to non-COVID-19 related underlying conditions. R002 was the only case to receive concurrent treatment with dexamethasone and remdesivir, from day 2 to 6 following sotrovimab. The S:E340K mutation was detected on Day 13 which corresponded to peak viral load for this case. In contrast, a resistance mutation was detected at day 7 for R003 followed by a gradual decline in viral load without additional COVID-19 treatment. 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 December 21, 2021. ; https://doi.org/10.1101/2021.12.18.21267628 doi: medRxiv preprint RNA extracted from specimens collected from case R004 underwent confirmatory metagenomic sequencing, which removes bias from SARS-CoV-2 RNA amplification.

Between 49 and 577 million reads were generated per specimen which were collected 2, 6, 11 and 15 days after sotrovimab. These reads produced consensus SARS-CoV-2 genomes with >99.9% coverage with 579-55,767X average depth. The results confirmed the presence of the S:E340K mutation six and 11 days after sotrovimab, but was not detected 15 days postinfusion.

Household contacts on R001 who became SARS-CoV-2 RT-PCR positive two days after R001, did not harbour mutations that confer resistance to sotrovimab. Table S3) .

Of the 527,931 international SAR-CoV-2 genomes 130, 101 and 24 contained the mutation E340K/A/V, respectively. A further 65 genomes contained the mutation S:P337L.

The overall prevalence of these mutations was initially very low in March 2020, however the proportion of international sequences carrying all three mutations increased by September 2021 (Supplementary Figures S4-7) . 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 December 21, 2021. ;  emerge. These individuals may receive a combination of new COVID-19 therapies to aid viral clearance and prevent severe disease. As numerous in vitro and clinical case studies have highlighted, close monitoring of these infections is warranted to ensure viral clearance and to detect the development of variants that have the ability to evade emerging therapeutic options. 10, [31] [32] [33] [34] [35] [36] [37] Our findings demonstrated the acquisition of mutations in the RBD of the SARS-CoV-2 S protein 6-13 days after treatment with sotrovimab. The genomic position of these mutations has previously been suggested to decrease the ability of sotrovimab to neutralize SARS-CoV-2 by 10-297-fold. 4 The most commonly detected mutation E340K (3 of 4 cases) was reported to confer the highest levels of resistance.

Previous in vitro studies have demonstrated that RBD mutations can lead to reductions in the effectiveness of mAbs and natural or vaccine-elicited neutralizing antibodies. 10, 34 This study adds important clinical data to support the experimental evidence. The mutations described were within highly conserved epitopes of SARS-CoV-2 with <250 international genomes reporting S:E340K/A/V changes in the spike protein, and this mutation has not occurred in VOC Alpha, Beta, Gamma and Delta. 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 December 21, 2021. ; https://doi.org/10.1101/2021.12.18.21267628 doi: medRxiv preprint resolution to detect mutations that develop following COVID-19 treatments and to explain the mechanism of breakthrough infections. The recent observation of G339D mutation in spike in the rapidly spreading Omicron VOC is also of concern. 39 Therefore, the incidence of these mutations in patients treated with sotrovimab may be underestimated.

In conclusion, SARS-CoV-2 genome analysis and culture in patients treated with sotrovimab can assist in monitoring the progress of COVID-19 infection and managing infection control and duration of isolation periods. Post-marketing genomic surveillance of patients that receive monoclonal antibody therapy for SARS-CoV-2 is prudent to minimize the risk of treatment failure, and transmission of potentially more resistant SARS-CoV-2 variants in both healthcare settings and the community.

Fastq files have been deposited in BioProject PRJNA633948 for all 50 genomes produced in this study. Individual SRA and Global initiative on sharing all influenza data (GISAID) accessions are available in Supplementary Data S1. 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 December 21, 2021. ; https://doi.org/10.1101/2021.12.18.21267628 doi: medRxiv preprint shows the SARS-CoV-2 viral load at each sampling point (right-hand y-axis scale. All cases were hospitalized during sampling periods.

. 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

The copyright holder for this this version posted December 21, 2021. ; https://doi.org/10.1101/2021.12.18.21267628 doi: medRxiv preprint

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The authors are grateful to NSW Pathology partner laboratories, ACT Pathology, Douglass