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A potentially disturbing new study published in December 2020 on the bioRxiv * preprint server suggests that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is causing the current 2019 coronavirus disease pandemic (COVID-19) may undergo strategic mutations that affect the immune capacity of the host to recognize and combat the pathogen via T effector cells.
Broad immune response
The virus triggers a wide spectrum of immune responses, both innate and adaptive. CD8 + cytotoxic T lymphocyte (CTL) responses occur in these patients, in response to recognition of a number of antigenic epitopes. CTLs are very important in clearing the infection because they kill the cell infected with the virus.
This action is triggered by the recognition of the viral peptides displayed on the surface of the host cell, after their specific presentation by the correct human leukocyte antigen (HLA) in order to activate the corresponding CTLs. The group of HLA antigens is produced by genes encoding the major histocompatibility complex, class I (MHCI).
There is ample evidence that CTL control of some RNA viruses triggers the emergence of viral mutations that prevent MHC-1 restricted recognition of viral antigens with subsequent destruction by CTLs.
Study details
The current study aimed to understand the effect of mutations in SARS-CoV-2 on presentation of viral peptides via MHC-I. They used deep sequencing methods with the viral genome and bioinformatics to analyze the results. The genomes were from viral isolates from 747 patient samples.
The researchers examined 27 CTL epitopes that were shown to be presented by the common subtype HLA-A * 02: 01, having an allele frequency of 0.29 in Austria, and by the minor subtype HLA- B * 40: 01 (allele frequency 0.03-0.05 in Austria).
They found around 200 mutations that resulted in amino acid substitutions at the CTL epitopes, all present at frequencies of 0.02 or higher, in around 230 samples. The frequencies of 33 of them ranged from 0.1 to 0.5. Nine mutations had become the default allele in 53 samples from different patients. Some of the epitopes overlapped, giving 207 different epitopes. Of these, 27 were on anchor residues, while helper residues were affected by 14 mutations. These two types of amino acids are essential for the presentation of the MHC-I peptide.
Independent emergence of mutations
Several variants were found that emerged independently in different individuals after infection.
Researchers examined fixed mutations in more than 145,000 sequences retrieved from the Global Influenza Data Sharing Initiative (GISAID) database. This aggregate data set showed mutations in up to 7.34% of the epitopes. Each of the 27 CTL epitopes had 10-11,700 non-synonymous mutations, the average being 807. The low frequency mutations identified in the current analysis were also found in GISAID as fixed mutations.
An alanine to valine mutation in an epitope was found in more than 75 of the sequences analyzed. This particular allele was first reported in June 2020, but is now a defining mutation of the 20A.EU1 subclade. However, it was found to be present in samples taken from March to April, albeit at low frequency. This supports the emergence of the same mutation independently in different individuals.
Interestingly, longitudinal sampling of the same patients showed that mutant epitopes appeared at a later period of infection. This suggests that the positive selection pressure due to CTL effector activity shaped these mutations.
Lower binding strength for CTL epitopes
Modeling studies to estimate the binding strength of peptides in wild-type and HLA-A * 02: 01 and HLA-B * 40: 01 mutant viruses showed weaker peptide binding to MHC-I.
The researchers selected 11 and 17 wild-type and mutant peptides, respectively, that would have weaker binding strength, testing them against recombinant HLA-A * 02:01 or HLA-B * 40:01 proteins. They found that the wild-type 9/11 peptides bound to these HLA antigens, stabilizing their structure by strong binding at physiological temperature.
Of the mutants, however, 11 had reduced binding and stabilizing capacity for MHC-I. MEVTPSGTWL is a peptide that binds only to the HLA-B * 40:01 minor allele and not to HLA-A * 02:01. Other mutants showed weak or no binding, respectively. One of the predicted CTL epitopes was not bound by wild type or mutant peptides.
Mutations cause MHC-I to degrade
The researchers then constructed tetramers loaded with peptides of both HLA antigens, for wild-type and mutant peptides. They found that the latter type of tetramer binds to related T cells in response to T cell receptor activation at 4 ° C but not at 37 ° C. The most likely reason was loss of peptides leading to the disruption of the MHC-I structure.
Mutations weaken the cellular immune response
They also looked at peptide-specific effector cell responses in peripheral blood mononuclear cells (PBMCs), obtained from patients with COVID-19 with one of these alleles. Stimulation of HLA-matched PBMCs with these peptides confirmed that they were T-cell epitopes. These virus-stimulated T cells showed interferon-gamma secretion. However, the mutant peptides reduced the immune response, with fewer positive tetrameric CTLs and lower IFN-gamma secretion.
What are the implications?
The SARS-CoV-2 ORF8 protein is hypothesized to reduce expression of MHC-I molecules on the host cell, but further studies are needed. The results of this current study show that effector T cells can give rise to mutations in SARS-CoV-2 that escape immune surveillance. “These results imply that mutations found in SARS-CoV-2 isolates promote immune leakage from HLA-dependent recognition by CTLs.”
The low frequencies of the non-synonymous mutations showed that they did not bind, possibly due to the short periods of infection with this virus compared to HIV or HCV. Additionally, the fact that there are many distinct HLA profiles within the population affects the spread of the virus, as for each individual there are different sets of CTL epitopes that are triggered by infection. Thus, different selection pressures have been exerted by these different subsets, which shape different viral mutational escapements. More work is needed to understand how mutations in a single epitope affect virus control.
Our results highlight the ability of SARS-CoV-2 to evade adaptive immune responses and provide further evidence for the impact of endogenous CTL responses and their participation in conferring protection in natural and vaccine-induced immunity. .
*Important Notice
bioRxiv publishes preliminary scientific reports which are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behaviors, or treated as established information.
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