A new potentially disturbing study published in December 2020 in bioRxiv * the prepress server suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the current pandemic of coronavirus disease 2019 (COVID-19) may suffer strategic mutations that affect the immune capacity of the host to recognize and fight the pathogen through T effector cells.
Wide immune response
The virus elicits a wide range of immune responses, both innate and adaptive. In these patients CD8 + cytotoxic T lymphocyte (CTL) responses occur in response to recognition of a number of antigenic epitopes. CTLs are very important in eliminating infection, as they kill the virus-infected cell.
This action is triggered by the recognition of viral peptides that are displayed on the surface of the host cell, after they are specifically presented by the appropriate human leukocyte antigen (HLA) to activate the corresponding CTLs. The HLA antigen group is produced by genes encoding the major class I histocompatibility complex (MHCI).
There is much evidence that CTL control of some RNA viruses causes the appearance of viral mutations that prevent restricted MHC-1 recognition of viral antigens with subsequent killing by CTLs.
Details of the study
The present study sought to understand the effect of SARS-CoV-2 mutations on viral peptide presentation by MHC-I. They used deep sequencing methods with the viral genome and bioinformatics to analyze the results. Genomes came from viral isolates from 747 patient samples.
The researchers examined 27 CTL epitopes shown to be presented by the common subtype HLA-A * 02: 01, with an allele frequency of 0.29 in Austria, and by the minor subtype HLA-B * 40: 01 (frequency of alleles 0.03- 0.05 in Austria).
They found about 200 mutations that resulted in amino acid substitutions in CTL epitopes, all present at frequencies of 0.02 or more, in about 230 samples. The frequencies of 33 of them were between 0.1 and 0.5. Nine mutations had become the default allele in 53 samples from different patients. Some of the epitopes overlapped, resulting in 207 different epitopes. Of these, 27 were in anchor residues, while auxiliary residues were affected by 14 mutations. These two types of amino acids are essential for the presentation of the MHC-I peptide.
Independent appearance of mutations
Several variants were found that had appeared independently in different individuals after infection.
The researchers examined fixed mutations in more than 145,000 sequences retrieved from the Global Initiative for Sharing All Influenza Data (GISAID) database. This global data set showed mutations in up to 7.34% of epitopes. Each of the 27 CTL epitopes had 10–11,700 non-synonymous mutations, with an average of 807. The low-frequency mutations identified in the current analysis were also found in GISAID as fixed mutations.
A mutation of alanine to valine was found in an epitope 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, this was found to be present in samples collected from March to April, albeit with low frequency. This supports the occurrence of the same mutation independently in different individuals.
Interestingly, longitudinal sampling of the same patients showed that mutant epitopes emerged in a later period of infection. This suggests that positive selection pressure due to CTL effector activity was shaping these mutations.
Weaker binding force for CTL epitopes
Modeling studies to estimate the binding strength of peptides in the wild-type and mutant viruses at HLA-A * 02: 01 and HLA-B * 40: 01 showed weaker binding of the peptide to MHC-I.
The researchers selected 11 and 17 wild-type and mutant peptides, respectively, that were predicted to have lower binding strength, testing them against recombinant HLA-A * 02: 01 or HLA-B * 40: 01 proteins. They found that 9/11 wild-type peptides bound to these HLA antigens, stabilizing their structure by strong binding at physiological temperature.
However, of the mutants, 11 had reduced binding and stabilization capacity by MHC-I. MEVTPSGTWL is a peptide that binds only to the minor allele HLA-B * 40: 01 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 mutant or wild-type peptides.
Mutations cause rupture of the MHC-I
The researchers constructed tetramers loaded with peptides from both HLA antigens, both for wild-type peptides and for mutants. They found that the latter type of tetramer bound to cognitive T cells in response to T cell receptor activation at 4 ° C but not at 37 ° C. The most likely reason was the loss of peptides that caused the MHC-I structure to break down.
Mutations weaken the cellular immune response
They also examined peptide-specific effector cell responses in peripheral blood mononuclear cells (PBMCs), obtained from COVID-19 patients with any of these alleles. Stimulation of HLA-combined PBMCs with these peptides confirmed that they were actual epitopes of T cells. These virus-stimulated T cells showed interferon-gamma secretion. However, the mutant peptides reduced the immune response, with fewer positive CTLs for the tetramer and lower IFN-gamma secretion.
What are the implications?
It is hypothesized that the SARS-CoV-2 ORF8 protein reduces the expression of MHC-I molecules in the host cell, but further study is required. The findings of this current study show that effector T cells can lead to SARS-CoV-2 mutations that escape immune surveillance. “These results imply that mutations found in SARS-CoV-2 isolates promote immune escape from HLA-dependent recognition by CTLs.”
The low frequencies of non-synonymous mutations showed that they had not been fixed, perhaps due to the short periods of infection with this virus compared to HIV or HCV. In addition, the fact that there are many separate HLA profiles in the population affects the spread of the virus, as for each individual there are different sets of CTL epitopes that are triggered by infection. Therefore, different selection pressures were exerted for these variable subsets, which form different viral mutational leaks. 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 elude adaptive immune responses and provide further evidence of the impact of endogenous CTL responses and their participation in the protection conference on natural and vaccine-induced immunity. “
* Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice / health-related behavior, or treated as established information.