In-depth analysis of SARS-CoV-2 quasispecies is pivotal for a thorough understating of its evolution during infection. The recent deployment of COVID-19 vaccines, which elicit protective anti-spike neutralizing antibodies, has stressed the importance of uncovering and characterizing SARS-CoV-2 variants with mutated spike proteins. Sequencing databases have allowed to follow the spread of SARS-CoV-2 variants that are circulating in the human population, and several experimental platforms were developed to study these variants. However, less is known about the SARS-CoV-2 variants that are developed in the respiratory system of the infected individual. To gain further insight on SARS-CoV-2 mutagenesis during natural infection, we preformed single-genome sequencing of SARS-CoV-2 isolated from nose-throat swabs of infected individuals. Interestingly, intra-host SARS-CoV-2 variants with mutated S genes or N genes were detected in all individuals who were analyzed. These intra-host variants were present in low frequencies in the swab samples and were rarely documented in current sequencing databases. Further examination of representative spike variants identified by our analysis showed that these variants have impaired infectivity capacity and that the mutated variants showed varied sensitivity to neutralization by convalescent plasma and to plasma from vaccinated individuals. Notably, analysis of the plasma neutralization activity against these variants showed that the L1197I mutation at the S2 subunit of the spike can affect the plasma neutralization activity. Together, these results suggest that SARS-CoV-2 intra-host variants should be further analyzed for a more thorough characterization of potential circulating variants. Author summary The global employment of mRNA-based COVID-19 vaccines, together with their high protection, has enabled to control the pandemic in places with high vaccination rate. However, the recent increase in the frequency of SARS-CoV-2 variants with reduced sensitivity to antibody neutralization has raised concerns about the ability of COVID-19 vaccines to eradicate SARS-CoV-2. Despite in-depth characterization of such variants, little is known about SARS-CoV-2 evolution within the host. Here, we preformed in-depth analysis of swab samples isolated from infected individuals and demonstrated that intra-host variants with reduced infectivity are developed during the course of the infection. Furthermore, these variants have varied sensitivity to the BNT162b2 vaccine and to convalescent plasma. In addition, we identified a point mutation at the S2 subunit of the spike protein, which impairs antibody neutralization activity. Our findings shed light on SARS-CoV-2 adaptation to the human host and highlight the importance of mapping the landscape of SARS-CoV-2 intra-host variability for better evaluating variants of concern.
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