key: cord-0684742-bpv5wgm5
authors: Chen, L.; Zody, M. C.; Mediavilla, J. R.; Cunningham, M. H.; Composto, K.; Chow, K. F.; Kordalewska, M.; Corvelo, A.; Oschwald, D. M.; Fennessey, S.; Zetkulic, M.; Dar, S.; Kramer, Y.; Mathema, B.; Maniatis, T.; Perlin, D. S.; Kreiswirth, B. N.
title: Emergence of multiple SARS-CoV-2 antibody escape variants in an immunocompromised host undergoing convalescent plasma treatment
date: 2021-04-11
journal: nan
DOI: 10.1101/2021.04.08.21254791
sha: 05afe10ea9f4d8fadf6350017d743b3f29ba626b
doc_id: 684742
cord_uid: bpv5wgm5

SARS-CoV-2 Variants of Concerns (VOC), e.g., B.1.351 (20H/501Y.V2) and P1 (20J/501Y.V3), harboring N-terminal domain (NTD) or the receptor-binding domain (RBD) (e.g., E484K) mutations, exhibit reduced in vitro susceptibility to convalescent serum, commercial antibody cocktails, and vaccine neutralization, and have been associated with reinfection. The accumulation of these mutations could be the consequence of intra-host viral evolution due to prolonged infection in immunocompromised hosts. In this study, we document the microevolution of SARS-CoV-2 recovered from sequential tracheal aspirates from an immunosuppressed patient on tacrolimus, steroids and convalescent plasma therapy, and identify the emergence of multiple NTD and RBD mutations associated with reduced antibody neutralization as early as three weeks after infection. SARS-CoV-2 genomes from the first swab (Day 0) and three tracheal aspirates (Day 7, 21 and 27) were compared at the sequence level. We identified five different S protein mutations at the NTD or RBD regions from the second tracheal aspirate sample (21 Day). The S:Q493R substitution and S:243-244LA deletion had ~70% frequency, while ORF1a:A138T, S:141-144LGVY deletion, S:E484K and S:Q493K substitutions demonstrated ~30%, ~30%, ~20% and ~10% mutation frequency, respectively. However, the third tracheal aspirate sample collected one week later (Day 27) was predominated by the haplotype of ORF1a:A138T, S:141-144LGVY deletion and S:E484K (> 95% mutation frequency). Notably, S protein deletions (141-144LGVY and 243-244LA deletions in NTD region) and substitutions (Q493K/R and E484K in the RBD region) previously showed reduced susceptibly to monoclonal antibody or convalescent plasma. The observation supports the hypothesis that VOCs can independently arise and that immunocompromised patients on convalescent plasma therapy are potential breeding grounds for immune-escape mutants.

Introduction and bcftools v1.11 9 . SNP and InDels were called using FreeBayes v1.3.5 1 0 0 (https://github.com/freebayes), followed by annotation using SnpEff v4.5 10 . A minimum variant 1 0 1 calling frequency was set to be 5% to identify within host variations. 1 0 2 1 0 3

The resulting SARS-CoV-2 viral genome sequences were uploaded to Nextclade server 1 0 4 (https://clades.nextstrain.org/) to assign Nextstrain clades 11 . SARS-CoV-2 lineage was 1 0 5 determined using Pangolin v2.3.0 (https://github.com/cov-lineages/pangolin) and GISAID clade 1 0 6 is determined based upon the clade specific marker variants from https://www.gisaid.org 12 . In 1 0 7 addition, 2,282 SARS-CoV-2 genomes with E484K mutation were downloaded from GISAID 1 0 8 database 12 (date as 2/12/2021), and the genomes with less than 1% ambiguous nucleotides 1 0 9

(Ns) and > 28,900 bp were aligned using MAFFT v7.475 12 using default setting. A maximum 1 1 0 likelihood phylogenetic tree was constructed using IQ-TREE v2.1.2 13 with automatic model 1 1 1 selection and 1000-bootstrap replicates. The resulting tree was annotated using ITOL v5 14 . A male in early 50s presented to a Northern Jersey hospital with fever, productive cough, 1 2 0 generalized myalgias, and progressive shortness of breath for 4 days (Fig1A). He had history of 1 2 1 deceased donor kidney transplant for end-stage renal disease (ESRD) secondary to HTN, 1 2 2 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 11, 2021. ; https://doi.org/10.1101/2021.04.08.21254791 doi: medRxiv preprint 1 3 6

He was treated with high-titer convalescent plasma (Day 1) and tocilizumab (Day 2). Due to his 1 3 7 worsening respiratory status, the patient was intubated (Day 2). Antibiotics were switched to 1 3 8 vancomycin and piperacillin-tazobactam and then discontinued as the patient was afebrile (Day 1 3 9

3). The patient was found to have bilateral deep venous thrombosis and was started on 1 4 0 therapeutic heparin (Day 3). Due to worsening hypoxic respiratory failure despite complete 1 4 1 support from mechanical ventilation, the patient was subsequently cannulated and placed on 1 4 2 veno-venous extra-corporeal oxygenation (ECMO) (Day 5). He went into rapid atrial flutter as 1 4 3 well and was started on intravenous amiodarone (Day 5). His renal failure attributed to multiple 1 4 4 factors such as his tacrolimus, COVID-19 injury, and hypotension slowly began to improve. 1 4 5

Oxygenation began to improve and stabilize, leading to tracheostomy (Day 16) and ECMO 1 4 6 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. vasopressors (Day 21). Following the antibiogram, antibiotics were de-escalated to ampicillin 1 5 0 (Day 21) and continued for a 7-day course. The septic shock resolved, and the patient was re-1 5 1 started on his anti-hypertensives once his blood pressure began to remain stable. His course 1 5 2 continued to be complicated by periodic desaturations and wide and narrow complex 1 5 3 tachycardia, anemia, and thrombocytopenia. He slowly improved permitting ventilation and 1 5 4 sedation weaning. As his dysphagia was unresolved during his recovery, percutaneous 1 5 5 endoscopic gastrostomy (PEG) tube was placed (Day 28) to improve nutritional status. He was 1 5 6 transferred to the step-down unit as he continued to recover (Day 49) and was discharged to a 1 5 7 long-term care facility (Day 64) requiring ventilatory support only at night. Unexpectedly, the 1 5 8 patient expired presumably due to hypoxic respiratory failure secondary to his COVID-19 1 5 9 pneumonia (Day 94). 1 6 0 1 6 1 Genomic analysis 1 6 2 SARS-CoV-2 positive qRT-PCR results ( Table 1) were obtained from three nasopharyngeal 1 6 3 swab samples (on Day 0, 34 and 41) and three tracheal aspirates (on Day 7, 21 and 27); the 1 6 4 first swab and the three tracheal aspirates were available for viral genome sequencing (Fig1A).

The genotype of the initial swab and tracheal aspirate (Day 7) were identical. The genomes of 1 6 6 these two samples harbored 14 mutations (versus Wuhan-Hu-1), and were assigned as 1 6 7

Nextstrain clade 20C, Pangolin lineage B.1.369 and GISAID clade GH, distinct from the three 1 6 8 501Y VOCs (Fig1B, C) . The second tracheal aspirate specimen (from Day 21) showed five 1 6 9 different S protein mutations at the NTD or RBD regions. The S protein Q493R substitution and 1 7 0 243-244LA deletion had ~70% frequency, while ORF1a A138T, S protein 141-144LGVY 1 7 1 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 11, 2021. ; https://doi.org/10.1101/2021.04.08.21254791 doi: medRxiv preprint

The Sequence Alignment/Map format and 10

A program for annotating and predicting the