Study: SARS-CoV-2 variants do not evolve to promote further escape from MHC-I recognition. Image Credit: NIAID

SARS-CoV-2 variants do not evolve to escape T cell-mediated immunity

In a recent study published on bioRxiv*preprint server, researchers have demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants do not further evolve to evade T cell-mediated immunity.

Study: SARS-CoV-2 variants do not evolve to promote further evasion of MHC-I recognition. ​​​​​​Image credit: NIAID

Studies have demonstrated that mutations in the SARS-CoV-2 open reading frame 8 (ORF8) gene play a key role in modulating its pathogenesis and host adaptation by regulating levels of the major complex d histocompatibility class I (MHC-I) and interferon-stimulated (ISG) genes. Notably, the ORF8 protein induces autophagic degradation of MHC-I and confers resistance to cytotoxic T lymphocyte (CTL) surveillance. Now, during the early phase of the coronavirus disease 2019 (COVID-19) pandemic, studies have identified the rapid evolution of the SARS-CoV-2 ORF8 gene.

Several unique mutations within the ORF8 gene have been identified in SARS-CoV-2 (VOC) variants of concern; however, none increased the ability of ORF8 to suppress MHC-I expression. MHC-I induces antigen presentation is crucial for the activation of the 8 (CD8+) CTL differentiation cluster, which kills virus-infected cells. Subsequently, the researchers hypothesized that the VOC and its ORF8 gene evolved further to arrest MHC-I by evading the memory CD8+ T cell-mediated immunity established by prior infection or vaccination.

About the study

In the current study, researchers infected Calu-3 cells with SARS-CoV-2 variants and the ancestral strain (USA-WA1) to examine the transcriptional levels of MHC-I genes, which differ for each variant of the SARS-CoV-2. They also performed multiple sequence alignment of ORF8 protein sequences from SARS-CoV-2 variants to observe non-synonymous mutations.

In addition, the researchers investigated the prevalence of mutations in SARS-CoV-2 variants, for which they downloaded 3,059 SARS-CoV-2 genome sequence data from the initiative’s database. Global Avian Influenza Data (GISAID). The team also constructed six ORF8 mutants from SARS-CoV-2 variants to observe the variant-specific effect on the surface expression levels of MHC-I of infected cells.

Finally, the researchers performed live experiments in which they infected C57BL/6J mice intranasally with a mouse-adapted (MA) strain of SARS-CoV-2 to analyze lung epithelial cell MHC-I expression levels two days after infection.

Study results

Consistent with previous reports, USA-WA1 strain significantly downregulated human leukocyte antigen (HLA)-A, B, and C genes. Similarly, COV alpha and beta showed similar reduction in the expression of messenger ribonucleic acid (mRNA) HLA-A, B and C such as the USA-WA1 strain.

Single mutations are found in ORF8 gene of SARS-CoV-2 variants (A) Proportion of mutants in ORF8 genes of SARS-CoV-2 variants shown.  The indicated amino acid positions are selected based on the results of the multiple sequence alignment performed in Fig.S1.  The number of sequences analyzed for each line is indicated above each graph.  (B) Frequency of amino acids at enriched positions for mutants in each variant.  Amino acids shown in gray correspond to WT.  (C) Schematic diagram of ORF8 proteins of SARS-CoV-2 variants.  (D and E) HEK293T cells were transfected with plasmids encoding C-terminally tagged SARS-CoV ORF8a/b, SARS-CoV-2 ORF8 WT or SARS-CoV-2 ORF8 variants.  Forty-eight hours after transfection, cells were harvested and analyzed for cell surface HLA-ABC expression.  Data are presented as raw MFI (D) or as the ratio of MFI in Flag+ cells to Flag- cells normalized to SARS-CoV ORF8a value (E) (n=3).  Data are mean ± sd Data are representative of three independent experiments.  ***, p< 0,001

Single mutations are found in ORF8 gene of SARS-CoV-2 variants (A) Proportion of mutants in ORF8 genes of SARS-CoV-2 variants shown. The indicated amino acid positions are selected based on the results of the multiple sequence alignment performed in Fig.S1. The number of sequences analyzed for each line is indicated above each graph. (B) Frequency of amino acids at enriched positions for mutants in each variant. Amino acids shown in gray correspond to WT. (C) Schematic diagram of ORF8 proteins of SARS-CoV-2 variants. (D and E) HEK293T cells were transfected with plasmids encoding C-terminally tagged SARS-CoV ORF8a/b, SARS-CoV-2 ORF8 WT or SARS-CoV-2 ORF8 variants. Forty-eight hours after transfection, cells were harvested and analyzed for cell surface HLA-ABC expression. Data are presented as raw MFI (D) or as the ratio of MFI in Flag+ cells to Flag- cells normalized to SARS-CoV ORF8a value (E) (n=3). Data are mean ± sd Data are representative of three independent experiments. ***, p<0.001

Conversely, the Iota and Gamma variants showed weaker downregulation and upregulation of HLA class I genes, respectively. Overall, a majority of SARS-CoV-2 variants have reduced HLA transcription just like the ancestral virus. This raised the possibility that many ISG-induced pathways of MHC-I processing and presentation are attenuated in cells infected with Gamma and B.1.429.

Regarding mutations, the authors observed seven non-synonymous mutations and two deletions in the 10 SARS-CoV-2 variants examined. Additionally, they identified a premature stop codon at amino acid position Q27 of the Alpha VOC, which truncated the length of the ORF8 polypeptide and likely altered its functionality. However, the R52I and Y73C downstream mutations did not impact the Alpha ORF8 protein.

With the exception of Alpha and Omicron, all other SARS-CoV-2 COVs, including Beta, Gamma and Delta, harbored mutations or deletions in the ORF8 protein. Although none of these mutations were conserved among different lineages, the SARS-CoV-2 variants of interest, Epsilon and Iota, exhibited V100L and T11I mutations, respectively.

Additionally, the authors found several unique lineage-specific mutations. For example, the ORF8 L84S mutation identified in the SARS-CoV-2 clade S was unique and not seen in any other variant. Many mutations discovered via multiple sequence alignment analyzes were generally widespread in proportions ranging from 12.5 to 100%. These results indicated that lineage-specific mutations were acquired independently during the evolution of SARS-CoV-2, and that mutations in a particular amino acid were exclusive to a single lineage. Unlike the influenza A virus, SARS-CoV-2 completely stopped the induction of MHC-I in infected cells alive.

conclusion

Presumably, SARS-CoV-2, like many other viruses, has evolved ways to avoid efficient MHC-I-mediated antigen presentation to CD8+ T cellsbecause they play a vital role in the adaptive immune response of the host.

After investigating whether SARS-CoV-2 variants shut down the MHC-I host system, the authors found that this was not the case, the ability to reduce MHC-I expression remained unchanged throughout. along the evolution of VOCs. Conversely, SARS-CoV-2 COVs have evolved to limit the host’s type I interferon response. Therefore, it is highly likely that the ancestral strain of SARS-CoV-2 was fully optimized to evade CD8+ T cell-mediated immunity and did not experience any evolutionary pressure to further optimize its immune evasion strategy. .

This finding also raised the possibility that SARS-CoV-2 uses multiple redundant mechanisms to suppress MHC-I expression to ensure evasion of CTL destruction. Moreover, unlike influenza A or the Epstein-Barr virus, it alters the priming of CD8+ T lymphocytes.

Taken together, the study data demonstrated the ability of SARS-CoV-2 to potently avoid MHC-I-mediated antigen presentation to CD8+ T cells and the role of ORF8 in its replication and efficient transmission.

*Important Notice

bioRxiv publishes preliminary scientific reports that 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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