Year-long COVID-19 infection reveals within-host evolution of SARS-CoV-2 in a patient with B cell depletion

Background: B-cell depleting therapies may lead to protracted disease and prolonged viral shedding in individuals infected with SARS-CoV-2. Viral persistence in the setting of immunosuppression raises concern for viral evolution. Methods: Amplification of sub-genomic transcripts for the E gene (sgE) was done on nasopharyngeal samples over the course of 355 days in a patient infected with SARS-CoV-2 who had previously undergone CAR T cell therapy and had persistently positive SARS-CoV-2 nasopharyngeal swabs. Whole genome sequencing was performed on samples from the patient’s original presentation and 10 months later. Results: Over the course of almost a year, the virus accumulated a unique in-frame deletion in the amino-terminal domain of the spike protein, and complete deletion of ORF7b and ORF8, the first report of its kind in an immunocompromised patient. Also, minority variants that were identified in the early samples—reflecting the heterogeneity of the initial infection—were found to be fixed late in the infection. Remdesivir and high-titer convalescent plasma treatment were given, and the infection was eventually cleared after 335 days of infection. Conclusions: The unique viral mutations found in this study highlight the importance of analysing viral evolution in protracted SARS-CoV-2 infection, especially in immunosuppressed hosts, and the implication of these mutations in the emergence of viral variants.


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Individuals receiving B-cell depleting therapies can have protracted disease and prolonged viral 23 shedding [3][4][5][6]. Persistent shedding of viral RNA for weeks to months after onset of symptoms 24 has been reported, however viable virus is not detected after 9 days post illness onset in most 25 patients [7]. In contrast, viral replication has been detected in immunocompromised patients for 26 several months after initial infection [5,8,9]. Persistent viral replication in these patients is likely 27 the result of profound lymphocyte defects due to B-and T-cell depleting therapies or underlying 28 hematologic disease. Viral persistence in the setting of immunosuppression has raised concern 29 for viral evolution [9] and the emergence of variants, especially during treatment with convalescent 30 plasma [6].

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Recent studies have demonstrated that SARS-CoV-2 in immunocompromised hosts is 32 prone to significant deletion mutations in the spike protein, especially in the S1 region [5,8,9].  This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Case presentation -A woman in her 40's with type II diabetes mellitus and diffuse large B-cell 88 lymphoma (DLBCL), who had been in complete remission for three years, presented with fever, 89 headache, nasal congestion, and productive cough. She reported that her symptoms developed  This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The patient was discharged a month later with temperatures of 99-100°F, episodes of 114 worsening cough and 3L NC supplemental oxygen. Testing for SARS-CoV-2 by PCR on NP 115 swabs was performed monthly for 3 months and every 3 months, thereafter. These were positive 116 intermittently with Ct values ranging between 37 and 38 ( Fig. 1). Due to the patient's overall mild 117 to absent symptoms, positive SARS-CoV-2 tests during this period were thought to probably 118 reflect shedding of non-viable virus particles; however, repeat chest CTs over the same period 119 showed bilateral increasing multifocal ground-glass opacities with crazy paving pattern and mixed This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (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 October 5, 2021. ; https://doi.org/10.1101/2021.10.02.21264267 doi: medRxiv preprint background samples from Maryland from the time of the first to the second presentation using the 151 publicly available Nextstrain software package [18]. All samples from this patient clustered on the 152 same branch of the tree, with no intermixed background samples, indicative of a prolonged 153 infection over 335 days, rather than a re-infection event (Fig. 2).

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The original sample, taken on Day 1, contained 11 consensus changes from the 155 Wuhan/Hu-1 strain (NC_045512.2). To visualize evolution of the virus over time within the patient,

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we compared the consensus nucleotides in the later 6 samples to that of the first sample (Day 1).

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Other samples collected the first month of infection had between 1-5 consensus changes 158 compared to the initial sample, whereas the later samples (Days 313-331) had 28 (Day 313) and 159 26 (Day 314) consensus changes at the nucleotide level from the initial sample. Of those, 19 and 160 17, respectively, were non-synonymous, with four substitutions in the spike protein (Fig. 3A).

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More interestingly, the later samples contained 2 deletions: a gap at nt 22290 to 22298 that led 162 to a unique del244-246 and, consequently, a A243G substitution (Fig. 3B); and a 497nt deletion 163 spanning the entire length of the ORF7b coding region and all but two amino acids of the ORF8 164 (Fig. 3C). Of note, several of the amino acid changes identified in the later samples (days 313 165 and 314) were present as minority variants in the initial samples, suggesting a heterogeneous 166 infection early on (Fig. 3D, smaller circles) with eventual fixation, as observed for ORF1a: 167 A3070V, ORF7a: S37F, and N: P365L. Conversely, a consensus change present in the early 168 samples also existed as minority variant in the last sample on Day 335 (Fig. 3D, smaller circles).

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The observed number of consensus changes in the later samples from the initial sample indicates  This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (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 October 5, 2021. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (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 October 5, 2021. ;https://doi.org/10.1101https://doi.org/10. /2021 identified in Singapore in January of 2020 [25,26]. In vitro analyses of similar deletions indicated 202 that deletion mutants had higher titers but similar levels of cytopathic effect and showed that the 203 virus was still transmissible. However, the deletion mutant may be less effective at establishing 204 infection in a new host due to loss of immune evasion features of ORF8 [26]. ORF8 has been 205 established as a key antagonist of innate immunity and has been shown to elicit a robust and 206 highly specific immune response during infection, suggesting that the deletion in competent hosts 207 may be due to immune driven selection [27]. Thus, it was surprising to see emergence of this 208 large deletion in our immunocompromised patient. It is possible that the immunocompromised 209 nature of this patient removes a need for ORF8 during infection. This hypothesis is supported by 210 data that show ORF8 is particularly tolerant to mutation, acquiring many missense and nonsense 211 mutations. ORF8 was also shown to be dispensable in cell culture [28]. A retrospective cohort 212 study performed on patients in Singapore found that some patients carried a mix of wild-type and 213 a 382nt deletion mutant in the ORF7-ORF8 region, while others only had the deletion mutant.

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Over time, the deletion mutant outcompeted the non-deletion virus [29]. In our study, we also 215 found evidence of a mixed infection with deletion and non-deletion mutants in the Day 313 sample, 216 but infection with only the deletion mutant on Day 314, indicating that the same competition may 217 have occurred in this patient with rapid clearance of the wild-type virus (Fig. 3C).

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This case demonstrates that severely immunocompromised patients may experience  This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (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 October 5, 2021. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (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 October 5, 2021. ;https://doi.org/10.1101https://doi.org/10. /2021    This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (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 October 5, 2021. ; https://doi.org/10.1101/2021.10.02.21264267 doi: medRxiv preprint This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  L293F  T814I  A3070V  L3606F  A4067T  L3915F  K1883T  S50L  A653V  I1115L  T30I I121L E121A This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.