Cumulative SARS-CoV-2 mutations and corresponding changes in immunity in an immunocompromised patient indicate viral evolution within the host

Abstract

Different scenarios explaining the emergence of novel variants of concern (VOC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported, including their evolution in scarcely monitored populations, in animals as alternative hosts, or in immunocompromised individuals. Here we report SARS-CoV-2 immune escape mutations over a period of seven months in an immunocompromised patient with prolonged viral shedding. Signs of infection, viral shedding and mutation events are periodically analyzed using RT-PCR and next-generation sequencing based on naso-pharyngeal swabs, with the results complemented by immunological diagnostics to determine humoral and T cell immune responses. Throughout the infection course, 17 non-synonymous intra-host mutations are noted, with 15 (88.2%) having been previously described as prominent immune escape mutations (S:E484K, S:D950N, S:P681H, S:N501Y, S:del(9), N:S235F and S:H655Y) in VOCs. The high frequency of these non-synonymous mutations is consistent with multiple events of convergent evolution. Thus, our results suggest that specific mutations in the SARS-CoV-2 genome may represent positions with a fitness advantage, and may serve as targets in future vaccine and therapeutics development for COVID-19.

Fig. 1. Timeline of the prolonged SARS-CoV-2 infection and viral load.

Timeline of the course of disease in an immunocompromised female patient with almost continuous viral shedding throughout the study period of 207 days. Ct, cycle threshold; IVIG therapy, intravenous immunoglobuline therapy; RTX, rituximab and bendamustine therapy. Source data are provided as a Source Data file.

Fig. 2. Chronology of the evolution of intra-host mutations.

The emergence of intra-host mutations in an immunocompromised patient with adequate humoral and lacking cellular immune response. The study period comprised 140 days of almost permanent viral shedding. High-quality next-generation sequences could be obtained at 14 time-points during the seven-month study period (starting on day 73, ending on day 207 with the last SARS-CoV-2 positive swab) and disclosed the chronological development of mutational events of SARS-CoV-2 as an answer to a unilateral immune response with strong antibody answer but lack of specific T-cells. RTX therapy, rituximab therapy; IVIG therapy, intravenous immune globulin therapy; single-event, temporary mutation. Source data are provided as a Source Data file.

Fig. 3. Chronology and frequency of the appearance of convergent intra-host mutations.

Evolution of mutations in the region coding for spike in a strain of the clade B.1.1, as they have been proven in identical form in the VOC. α, mutations are described for the Alpha variant B.1.1.7; β, Beta variant B.1.351; δ, Gamma variant B.1.1.28.1; γ, Delta variant B.1.617.2; o, Omicron variant B.1.1.529. The red line graph shows the mean frequency of all mutations at a given day. The development of mutations did not occur linearly, but rather in a fluctuating pattern, with frequent replacement by the wildtype variant. Source data are provided as a Source Data file.

Fig. 4. Representation of all acquired mutations during prolonged infection.

The acquired and temporarily acquired mutations of the investigated strain in the course of a seven-month-long infection in an immunocompromised person. Overall, 17 persistent or temporary spike mutations were evolved, whereas nine (52.9%) turned out to be temporary and were subsequently replaced by the wild-type variant. *Temporary mutations, S1 spike 1, S2 spike 2, hr heptad repeat, RBD receptor binding domain. The mutations marked in orange are also found in the Omicron variant (B.1.1.529) in similar or identical expression (10 out of 17), mutations marked in red are found in other VOC (3 of 17; 17.6%). All acquired mutations occurred in the regions ORF1a (n = 1), ORF1b (n = 1), ORF8 (1) and the spike (n = 17). Thirteen of the 17 mutations (76.5%) acquired in the course of the prolonged infectious phase are already described mutations in VOC. Source data are provided as a Source Data file.

Fig. 5. Outgroup-routed consensus tree.

The tree is based on 40 SARS-CoV-2 whole genome sequences with 29,806 nucleotide sites. The section highlighted in red shows the monophyletic clade of variants newly formed in an immunocompromised patient, embedded in prominent VOC and typical Austrian strains sequenced in the same investigation period, downloaded from GISAID and supplemented by early Austrian sequences of the Omicron variant in December 2021. Numbers at nodes indicate bootstrap support values (only values >50 are shown).