Viral Population Heterogeneity and Fluctuating Mutational Pattern during a Persistent SARS-CoV-2 Infection in an Immunocompromised Patient

The study investigates how SARS-CoV-2 evolves within a single, deeply immunocompromised host over a prolonged infection. Although SARS-CoV-2 has lower variability than many RNA viruses due to ExoN proofreading, the pandemic has shown ongoing emergence of variants with mutations that can increase infectivity and enable immune escape. Prior work implicates selective pressures from endogenous immunity and exogenous antibody therapies in driving viral evolution, particularly in immunocompromised individuals with hematologic malignancies. The authors present a patient with stage III-A follicular lymphoma and profound B-cell depletion who, despite two vaccine doses, experienced a severe SARS-CoV-2 infection with high-level viral shedding persisting over 7 months. The research question is whether sustained replication in such a host is accompanied by intra-host viral evolution that generates heterogeneous viral subpopulations, and how immune milieu and treatments correlate with this evolution. The purpose is to characterize longitudinal genomic changes and relate them to serologic findings, with implications for the emergence of variants of concern.

The introduction synthesizes evidence that selective pressures, including monoclonal antibodies and convalescent plasma, can drive SARS-CoV-2 evolution, demonstrated by in vitro culture under antibody pressure and by longitudinal studies of immunocompromised patients on B-cell–depleting therapies. Recurrent deletions in the spike N-terminal domain enable antibody escape and have been repeatedly observed in both patients and experimental systems. Prior case studies document prolonged infections in patients with lymphoid malignancies, with accumulation of numerous mutations including known escape and infectivity-enhancing substitutions that overlap with those in widely circulating variants. Epidemiological context in Italy at the turn of 2021–2022 is reviewed to assess reinfection versus reactivation likelihood, noting rapid replacement of Delta (B.1.617.2) by Omicron (B.1.1.529), supporting within-host persistence/reactivation rather than reinfection with a different lineage.

Design: Longitudinal case study of a single immunocompromised patient with persistent SARS-CoV-2 infection. Clinical case: 61-year-old male with stage III-A follicular lymphoma (BCL2+), prior CMV infection, double-vaccinated (BNT162b2). Received Bamlanivimab/Etesevimab on 7 Dec 2021, hospitalized 24 Dec for interstitial pneumonia, transiently PCR-negative on 28 Dec. Re-positive 4 Feb 2022; treated with Paxlovid 28 Feb to 4 Mar; later diagnosed with Pneumocystis jirovecii (treated with Atovaquone). Lymphocyte typing showed low CD4+/CD8+ ratio and B-cell depletion; started IVIG on 31 May, repeated every 21 days. Sampling and diagnostics: Nasopharyngeal swabs collected from 3 Dec 2021 to 9 Jul 2022; routine qRT-PCR (Xpert Xpress SARS-CoV-2) with N gene Ct as viral load proxy. Persistently positive except 28 Dec 2021. Eight samples collected between 14 Apr (Day 132) and 9 Jul 2022 (Day 218) were included for sequencing (Ct < 30 eligibility). Serology performed periodically from 27 Dec 2021: anti-Spike IgG (LIAISON SARS-CoV-2 Trimeric-S CLIA) peaked after mAb administration then declined to plateau by May–June 2022; anti-Nucleocapsid IgG (Abbott CMIA) undetectable throughout, indicating absent endogenous humoral response. RNA extraction and library prep: RNA extracted/purified with Maelstrom 9600 (TANBead). Libraries prepared using CleanPlex SARS-CoV-2 Flex Research and Surveillance NGS Panel (Paragon Genomics) per manufacturer, two-pool multiplex PCR workflow; indexing with Illumina i7/i5. Automation via Hamilton Microlab STAR. Sequencing: Libraries quantified (Qubit dsDNA HS), normalized to 10 nM, pooled to 4 nM, denatured/diluted to 1 pM, sequenced paired-end, dual-indexed on Illumina MiSeq (reagent kit v2) with 5% PhiX spike-in. Bioinformatics: Reads aligned to reference SARS-CoV-2 Wuhan-Hu-1 (NC_045512.2) using SOPHiA-DDM-v4 for consensus and variant calling with 70% frequency cutoff. Lineages assigned with Pangolin. Variants compared to reference B.1.617.2 AY.122 (OW998398.1) to distinguish lineage-defining versus intra-host-derived mutations. Ethics: Samples anonymized per local Ethical Board regulations (AVR-PPC P09, rev.2).

- All eight genomes belonged to B.1.617.2 AY.122 (Delta) lineage. - Evidence of a complex, dynamic intra-host viral population comprising three related subpopulations that alternated in dominance over time: • Subpopulation 1: Days 132, 152, 176; 14–15 variants (8 missense, 3 synonymous, 2–3 deletions, 1 non-coding) vs AY.122 reference. • Subpopulation 2: Days 144, 165; 15 variants (8 missense, 4 synonymous, 2 deletions, 1 non-coding). Subpops 1 and 2 shared 13 persistent mutations; 4 mutations fluctuated (temporary appearance followed by reversion or replacement). • Subpopulation 3 (late phase): Days 189, 200 (20 variants: 13 missense, 4 synonymous, 2 deletions, 1 non-coding) and Day 218 (26 variants: 19 missense, 4 synonymous, 2 deletions, 1 non-coding). Likely evolved from subpopulation 2 (15 shared mutations; 13 shared across all three subpops). - Apparent accelerated intra-host evolution: Using an expected rate of ~6×10^-4 mutations/genome/year, the observed rate between Days 132–218 (86 days) was ~3.5×10^-5 per site per day (reported as significantly higher than expected); over 218 days overall ~2×10^-5 (likely underestimated due to missing early data). - Mutational spectrum: 28 sites total; 14 in spike (50%), 9 in ORF1ab (32%). • Spike NTD deletions within RDR2 and RDR4: Leu141_Tyr144del; Leu242_Leu244del; Ala243_Leu244del – deletions known to disrupt neutralizing epitopes and confer immune escape. • RBD/other spike substitutions affecting receptor binding or escape: Lys417Asn; Asn501Thr; substitutions near S1/S2 and S2: Ala570Gly; Val635Ala (later replaced by Val635Gly); His655Tyr; Ala684Val; late S2 changes Phe1103Leu and Asp1153Tyr also observed. • Functional attribution: 8 mutations (~29%) associated with enhanced immune escape; 6 (~21%) associated with increased receptor engagement/infectivity. • Temporal behavior: Some escape mutations were stable (e.g., Leu141_Tyr144del, Asn501Thr, Ala570Gly); others transient or replaced (e.g., Val635Ala lost; Leu242_Leu244del swapped with Ala243_Leu244del). Several infectivity-enhancing mutations emerged later (K417N, V635G, H655Y, A684V). - ORF1ab mutations (stable across samples) in nsp2, nsp3, nsp12 (RdRp), nsp14 (N7-methyltransferase), nsp15 (endoRNase), nsp16 (2′-O-methyltransferase), potentially contributing to replication/host adaptation. - No mutations detected in 3CLpro (nsp5), Paxlovid’s target, suggesting limited antiviral-driven selection in this locus. - Clinical/serologic correlation: Persistently high viral loads (Ct values ~15–24 during study window) with absent anti-N IgG and waning anti-S IgG consistent with lack of endogenous humoral response and passive immunity from mAb administration; emergence of several spike escape mutations aligns temporally with early antibody pressure. - Epidemiology supports persistent infection/reactivation rather than reinfection: despite Delta’s rapid replacement by Omicron in Italy after Dec 2021, all sequences remained AY.122.

Sustained high-titre replication in a profoundly immunocompromised host facilitated ongoing intra-host evolution, generating a heterogeneous viral population with alternating sublineages. The temporal appearance and fluctuating frequencies of escape and infectivity-enhancing mutations suggest a dynamic interplay between viral replication and selective pressures from the host environment, including passive immunity from monoclonal antibodies. Early emergence and persistence of multiple spike NTD deletions and RBD/S2 substitutions known for immune escape imply exogenous antibody-driven selection, while later acquisition of mutations enhancing receptor binding and entry points to continued adaptation to the host niche. The dominance shift to a third subpopulation late in infection indicates selection and further divergence over time. Epidemiologic context and lineage consistency argue against reinfection and favor reactivation/persistence of the original AY.122 virus. The mutational patterns mirror changes seen in globally circulating variants, supporting the notion that prolonged infections in immunocompromised individuals can serve as incubators for antigenically novel variants with potential public health implications. These findings highlight the importance of monitoring such patients for both clinical management and genomic surveillance, as well as the need to understand how B-cell depletion and related cellular immunity defects influence within-host evolutionary trajectories.

This case report demonstrates that persistent SARS-CoV-2 infection in a severely immunocompromised patient is accompanied by accelerated intra-host evolution and diversification into genetically distinct subpopulations. Multiple spike mutations conferring immune escape and enhanced infectivity emerged, some stabilizing while others fluctuated, paralleling mutations observed in variants of concern. The study underscores immunocompromised hosts as potential reservoirs for the evolution of transmissible or immune-evasive variants, emphasizing the need for tailored clinical management and integrated genomic surveillance. Future directions include: expanding longitudinal cohorts of immunocompromised patients; integrating deeper intra-host frequency analysis below 70% thresholds; functional validation of observed mutations and combinations; evaluating the impact and timing of therapeutics (antivirals, monoclonal antibodies, IVIG) on evolutionary dynamics; and elucidating contributions of cellular immunity deficits to within-host selection.

- Single-patient case limits generalizability to broader immunocompromised populations. - Lack of genomic data from the first 132 days (earliest samples were discarded) impedes complete reconstruction of early evolutionary events and may underestimate mutation rates. - Study ended at Day 218, preventing follow-up on late-emerging mutations with intermediate frequencies. - No functional assays were performed to directly confirm phenotypic effects (escape, infectivity) of specific mutations or combinations in this patient context. - Variant calling threshold at 70% may miss lower-frequency intra-host variants and subpopulations.