Since its emergence in late 2019, SARS-CoV-2 has demonstrated an unprecedented adaptive capacity, enabling nearly unchecked human-to-human transmission and global spread. Despite its relatively lower genomic variability compared to other RNA viruses due to the proofreading activity of non-structural protein 14 (ExoN), SARS-CoV-2 evolution continues, with the periodic emergence of Variants Of Interest (VOI) and Variants Of Concern (VOC). VOCs carry mutations that can increase infectivity, reduce diagnostic and therapeutic effectiveness, or facilitate immune evasion, potentially ensuring continued circulation. Studies increasingly highlight the role of endogenous immune response and exogenous antibody administration (monoclonal antibodies or convalescent plasma) in shaping genomic diversity and driving viral evolution. In vitro studies and observations of immunocompromised patients with diminished or absent immune responses, often receiving immune treatments, reveal that persistent, uncontrolled viral replication can lead to an unusually high accumulation of mutations with potential biological and epidemiological significance. This study examines a deeply immunocompromised patient with stage III-A Follicular Lymphoma, underlying Cytomegalovirus and Pneumocystis jirovencii infections, who developed a persistent, high-load SARS-CoV-2 infection despite double vaccination. By monitoring accumulated viral mutations over seven months, coupled with serological screening to correlate immune response parameters with viral evolution, we aimed to characterize intra-host viral evolutionary dynamics and evaluate the potential of immunocompromised individuals as reservoirs for antigenically novel variants.

The introduction extensively reviews existing literature on SARS-CoV-2 evolution, highlighting the roles of immune response and antibody treatments in shaping viral diversity. It cites multiple studies showing the link between immunocompromised patients (particularly those with hematological malignancies and B-cell depleting therapies) and persistent, uncontrolled viral replication, characterized by an accumulation of mutations. The review emphasizes previous research illustrating the potential of such infections to generate variants with enhanced transmissibility or immune escape capabilities, making these patients potential reservoirs for viral evolution and spread. The literature reviewed underscores the need for further research to understand the mechanisms driving viral evolution in immunocompromised hosts and to assess the epidemiological implications.

This study followed a 61-year-old male patient diagnosed with stage III-A Follicular Lymphoma (BCL2+), treated with Rituximab-Bendamustine and Rituximab maintenance therapy. He also experienced Cytomegalovirus and Pneumocystis jirovencii infections and received two doses of the Comirnaty (BNT162b2) vaccine. His SARS-CoV-2 infection, initially detected on December 3, 2021, persisted for over seven months. Eight nasopharyngeal swabs were collected between April 14 and July 9, 2022, with qRT-PCR confirming persistent SARS-CoV-2 positivity (except for one negative result on December 28, 2021). Samples with N gene Ct values <30 were selected for next-generation sequencing (NGS). RNA extraction and purification were done using the Maelstrom 9600 system, and library preparation employed the CleanPlex SARS-CoV-2 Flex Research and Surveillance NGS Panel. Sequencing was performed on a MiSeq instrument (Illumina). Data analysis involved aligning sequenced reads to the SARS-CoV-2 Wuhan-Hu-1 reference genome using SOPHiA-DDM-v4, with variant calling using a 70% frequency cutoff. Lineage assignment was done using Pangolin. Mutations were compared against a reference B.1.617.2 AY.122 sequence to distinguish lineage-defining mutations from intra-host evolution. Serological tests monitored anti-Spike and anti-Nucleocapsid specific antibodies (IgG) to assess the correlation between immune response and viral evolution. Ethical approval was obtained from the local Ethical Board.

All eight genomic sequences belonged to the B.1.617.2 AY.122 lineage. Analysis revealed a structured, complex, and dynamic viral population. A chronology of persistent and temporary mutations was identified. The calculated mutation rate was significantly higher than expected, indicating accelerated intra-host viral evolution. Three genetically distinct, but related, subpopulations were identified, coexisting and exhibiting fluctuating dominance throughout the infection. Mutations predominantly affected the S gene (50%), followed by ORF1ab (32%). Several mutations conferred enhanced immune escape (29%) or increased receptor engagement (21%). Some escape mutations were stable, while others appeared and disappeared, sometimes replaced by the wild type or another mutation. Infectivity-enhancing mutations emerged later in the infection. The early emergence of several escape mutations suggests the influence of monoclonal antibody (Bamlanivimab/Etesevimab) administration. Mutations also affected non-structural proteins involved in the viral replication cycle. No mutations were found in the 3C-like protease (3CLpro), the target of Paxlovid. The study identified mutations that are also found in other circulating variants, suggesting convergent evolution. The study demonstrates a dynamic interplay between viral replication and host factors resulting in host adaptation.

This study showcases the impact of profound immunodeficiency on SARS-CoV-2 infection and evolution. The observed accelerated intra-host evolution with the emergence and fluctuation of multiple subpopulations demonstrates the virus's capacity to adapt within a single host. The early appearance of many immune escape mutations suggests a strong influence of monoclonal antibody treatment. The identification of mutations conferring both immune escape and increased infectivity reveals the virus's ability to evade the host's immune response while enhancing its ability to infect cells. The coexistence and dynamic shifts in the prevalence of distinct subpopulations highlight a complex interplay between the virus and the host immune system. These findings suggest that immunocompromised individuals may act as reservoirs for the development of new SARS-CoV-2 variants with altered characteristics.

This case study reveals a complex intra-host evolutionary pattern of SARS-CoV-2 in a severely immunocompromised patient. The findings highlight the importance of continuous monitoring of SARS-CoV-2 evolution in immunocompromised individuals and emphasize their potential role in generating new variants with significant epidemiological implications. Future research should focus on understanding the interplay between viral evolution, immune response, and treatment strategies in such patients.

The study's main limitation is its focus on a single patient, limiting the generalizability of the findings. The lack of genomic data from the early stages of infection (before April 14, 2022) may have underestimated the overall mutation rate and obscured the early dynamics of viral evolution. Further research involving a larger cohort of patients is needed to validate these observations and assess the broader significance of the observed mutations.