When and how do de novo spike mutations in SARS-CoV-2 emerge?

In a recent study published in transplant infectionresearchers evaluated viral mutations in two patients with persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Background

Mutations in SARS-CoV-2 are associated with enhanced contagiousness, immune escape, resistance to therapeutic agents, and impaired diagnostic detection. Several SARS-CoV-2 variants (VOCs) of concern share the same non-synonymous substitutions despite being from different lineages.

Understanding the mechanisms underlying the emergence of VOCs is important in developing strategies to prevent further spread. It has been hypothesized that the emergence of VOCs is due to the accumulation of genetic modifications that enhance viral fitness in persistently infected individuals.

About research

In this study, we described two immunosuppressed cases with prolonged SARS-CoV-2 infection. Remaining nasopharyngeal swabs and whole blood samples were obtained from each patient. Viral nucleic acids were isolated from nasopharyngeal samples and tested by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Whole-genome sequencing of viral RNA and phylogenetic and diversity analyzes were performed.

Spike N-terminal (NTD) and receptor binding (RBD) domain antibody titers were quantified using an enzyme-linked immunosorbent assay (ELISA). Standard curves were generated using anti-RBD (CR3022) and -NTD (SPD-M121) antibodies. Site-directed mutagenesis was performed using oligonucleotides containing the E484K/Q and del241-248 mutations.

We created an HIV-based luciferase reporter pseudovirus using a spike expression construct containing the E484K/Q substitution. Spike pseudotyped virus was used for cell entry and neutralization assays. HeLa-ACE2 cells were challenged with pseudovirus at multiple dilutions for cell entry assays. Neutralization assays were also performed, but cells were pre-incubated with serially diluted serum samples.

One 64-year-old patient (A) was diagnosed with mantle cell lymphoma in 2017 and underwent hematopoietic stem cell transplantation in August 2020. The patient received her first BNT162b2 vaccination in December 2020 and developed cough and fever 5 days later. The subject tested positive for her SARS-CoV-2 on 6 January 2021. The patient was subsequently hospitalized with radiological and clinical progression. The subject developed acute respiratory distress syndrome (ARDS) and died 7 months after diagnosis of coronavirus disease 2019 (COVID-19).

The second patient (B) is 48 years old and was diagnosed in March 2019 with chronic lymphocytic leukemia with Richter’s transformation. The patient tested positive for SARS-CoV-2 on 4 March 2021 and negative on 26 April 2021. However, mild symptoms persisted until June 2021 and required further evaluation. The patient was subsequently hospitalized on 25 June 2021, where she tested positive for SARS-CoV-2, and upon complete resolution of clinical symptoms, she was discharged on 4 July 2021. Patients were unvaccinated during the study period.

findings

Each patient had multiple tests for SARS-CoV-2 during hospitalization. Patient A was positive for SARS-CoV-2 on all 9 PCR tests over 130 days. Patient B. She tested positive twice (in 5 tests) in 117 days. Five positive specimens from patient A and two positive specimens from patient B were sequenced. Patient A’s consensus sequence mapped to clade 20B, and patient B’s consensus sequence mapped to her 20G.

Patient A had persistent symptoms and tested consistently positive for SARS-CoV-2 with the same 20B substrain each time. Patient B showed transient symptomatic improvement consistent with negative PCR results despite persistent respiratory symptoms. Similarly, the infection was the same her 20G substrain at each time point. Consensus sequences mapped to the same subphylegy, but Also Mutations emerged throughout the disease, and some later became dominant.

In addition, the team also observed that within-host diversity increased over time. 7 amino acid deletion (241-248) and 3 amino acid deletion (241-243) spike protein It was prevalent in patient A. Notably, the beta variant also contains the 241-243 deletion. Her E484Q substitution observed in the kappa variant was also found in the viral isolate from patient A. In contrast, a viral isolate from patient B developed her E484K substitution documented in beta and gamma variants.

Each patient received the monoclonal antibody bamuranivimab during initial treatment. Anti-NTD antibody was detected in patient A, but anti-RBD antibody was not detected. Patient B had no detectable antibodies to RBD or NTD. Since del241-243 and her E484K were previously associated with antibody escape, the authors determined whether other mutations detected in patient isolates displayed similar phenotypes.

They found that each mutation adversely affected viral fusion. E484Q/K substitutions caused minor effects, whereas deletions were associated with much greater dominant impairments.Neutralization assays using patient A serum samples showed that the double mutant pseudovirus (E484Q and del241-248) were much more resistant to neutralization than single mutants.

Conclusion

Observed studies Also Emergence of SARS-CoV-2 spike protein mutations in two immunosuppressed patients with prolonged SARS-CoV-2 infection. Spikes in blood Only one of her patients had antibodies to her NTD. Previously he had several mutations identified in VOCs. These mutations reduced cell entry efficiency and increased resistance to neutralization.

Altogether, these findings suggest that persistent infection in immunosuppressed hosts may apply selective pressure to improve viral fitness. Further studies are needed to characterize and predict the evolution of SARS-CoV-2.