COVID-19: World’s First HLA-I Immunopeptidome Study Of SARSCoV-2-Infected Human Cell Lines By Harvard University Reveals Alarming Details
: A Harvard led research on what can be termed as the world’s first detailed HLA-I immunopeptidomesStudy of SARSCoV-2-infected human cell lines while giving new insights into immune responses has also proven alarmingly details that the SARS-CoV-2 SARS-CoV-2 infection interferes with cellular pathways that may impact HLA-I peptide processing and presentation and immune signaling.
The study found that POMP (Proteasome Maturation Protein), a chaperone critical for the assembly of 20S proteasomes and immunoproteasomes, was significantly depleted in infected cell lines. POMP has recently been reported to impact ORF9c stability, which has been implicated in suppressing the antiviral response in cells (Dominguez Andres et al., 2020).
Several ubiquitination pathway proteins were significantly altered in response to SARS-CoV-2 infection, including depletion of TRIM26, an E3 ligase that modulates IRF3, NF-κB activation and IFN-β induction(Ran et al., 2016), and enrichment of TRIM7, which has recently been shown to bind M (Stukalov et al., 2020). Interestingly, the tyrosine kinase, JAK1, critical for IFN signaling that has been reported to be reduced with SARS-CoV nsp1 overexpression (Wathelet et al., 2007) was depleted in both cell lines upon SARS-CoV-2 infection.
Taken together, these data suggest that SARS-CoV-2 interferes with IFN signaling proteins and the HLA-I pathway through both POMP depletion and by altering ubiquitination enzymes, that in turn, may prevent highly expressed SARS proteins from being processed and presented.
The study findings are published on a preprint server and are currently being peer reviewed for journal publication. https://www.biorxiv.org/content/10.1101/2020.10.02.324145v1
The study findings could also improve investigation of T cell response to COVID-19.
The study team had made an important discovery about the processing and presentation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral peptides that provides a better understanding of the immune response to coronavirus disease 2019 (COVID-19) and may help facilitate the design of improved vaccines.
The researchers presented the first detailed analysis of viral peptides presented by class I human leukocyte antigen (HLA-I) on human cells infected with SARS-CoV-2.
Importantly, the study team found that nine of the viral peptides are derived from non-canonical open reading frames (ORFs) in the viral Spike protein and Nucleoprotein. Spike is the surface structure SARS-CoV-2 uses to bind to and infect host cells, while Nucleoprotein is involved in packaging viral RNA and the release of virions.
The researchers says the finding suggests that studies of the T cell response among COVID-19 patients that currently only focus on canonical viral ORFs exclude a significant number of virus-derived HLA-I epitopes.
Dr Pardis Sabeti , co-researcher from the Broad Institute of MIT and Harvard, says that the current pools of SARS-CoV-2 derived HLA-I peptides that enable T-cell activating viral signatures to be
identified may be incomplete.
Novel approaches that directly identify naturally presented SARS-CoV-2 peptides would help to inform the design of future pools to provide a more detailed characterization of T cell responses in COVID-19 patients. Such approaches would also facilitate the design of more effective vaccines, say the researchers.
Current research efforts to develop effective vaccines for SARS-CoV-2 must accept and understand the role that T cells play in establishing long-term immunity against the virus.
Following infection, viral proteins are processed and presented on the host cell surface by HLA-I for recognition by cytotoxic T cells, which then induce an immune response to combat the virus.
However some of the limitations to date is that most studies that have examined the interaction between T cells and SARS-CoV-2 antigens have used bioinformatic predictions of HLA-I binding affinity. However, this approach is limited because it does not account for all of the steps involved in antigen processing and presentation, and the average positive predictive values achieved across HLA alleles are still only about 64%.
Experimental design and measurements of HLA-I immunopeptidome, whole proteome and RNA-seq in SARS-CoV-2 infected cells. (A) Schematic representation of the experiment and the antigen presentation pathway. A549/ACE2/TMPRSS2 and HEK293T/ACE2/TMPRSS2 cells were infected with SARS-CoV-2 (Washington strain, accession number MN985325) at Multiplicity of Infection (MOI) of 3 in a BSL3 facility and harvested at 12, 18 and 24hpi (hours post infection). For HLA-I immunopeptidome measurements cells were lysed and HLA-I peptide complexes were immunoprecipitated. The flow-through after immunoprecipitation was collected for whole proteome measurements. For RNA-seq we added Trizol to infected cells, purified the RNA and performed strandspecific short reads sequencing. MS/MS spectra of immunopeptidome and proteome analysis were searched against a protein database including the human proteome, canonical and non-canonical SARS-CoV-2 ORFs and filtered at 1.5% FDR. (B) Population frequency of the 9 endogenous HLA-I alleles expressed in A549 and HEK293T cells. (C) Length distribution of HLA peptides in infected and naive cells. (D) Motif of 9-mer sequences identified in infected and naive cells. (E) Fraction of observed peptides assigned to alleles using HLAthena prediction (percentile rank cutoff <0.5) for immunopeptidome of infected and uninfected cells.
These prediction models also do not account for how viruses might alter cellular processes and affect antigen presentation.
Dr Sabeti added, “For example, viruses can attenuate translation of host proteins, downregulate the proteasome machinery, and interfere with HLA-I expression. These changes shape the collection of viral and human-derived HLA-I peptides presented to the immune system.”
He stressed that experimental measurements of the SARS-CoV-2 peptides presented on host cells are therefore, essential to understanding how the immune system responds to infection.
A better approach is to utilize mass spectrometry-based HLA-I immunopeptidomics as it is a direct and unbiased approach to discovering endogenously presented peptides and to establishing the processes that govern antigen processing and presentation.
Dr Sabeti told Thailand Medical News, “Leveraging HLA-I immunopeptidome datasets to learn virus-specific antigen processing rules will improve our ability to predict viral epitopes accurately and utilize them to study immune responses in COVID-19 patients.”
The detailed analysis of mass spectrometry-derived data requires selecting a set of viral ORFs to examine, and until now, such analyses have primarily been focused on canonical ORFs.
Significantly, over the last ten years, genome-wide sequencing studies have revealed a surprising number of non-canonical ORFs in viral genomes, the function of which remains largely unknown.
Key Findings Of This Current Study:
Dr Sabeti and colleagues have now presented the first detailed analysis of HLA-I immunopeptidome in two SARS-CoV-2-infected human cell lines using mass spectrometry, RNA-sequencing, and global proteomics measurements.
The study team identified viral HLA-I peptides derived not only from canonical ORFs but also from non-canonical ORFs in SARS-CoV-2 Spike and Nucleoprotein that are not captured by current vaccines.
Dr Sabeti added, “Remarkably, 9 of 29 viral peptides detected are derived from internal out-of-frame ORFs in S [Spike] (S.iORF1/2) and N [nucleoprotein] (ORF9b).”
Note that none of the current vaccines used in China or Russia or even those in phase 3 trials ever considered these.
The study team says this finding implies that current studies investigating T cell responses in COVID-19 patients that focus only on the canonical viral ORFs exclude a significant number of virus-derived HLA-I epitopes.
The study team estimated that based on the list of endogenously processed and presented SARS-CoV-2 peptides they identified, a pool of 24 peptides would provide one or more peptides for at least one HLA-I allele in 99% of the human population.
The researchers say the study findings could be used to facilitate the data-driven selection of peptides for immune monitoring and to improve the design of effective vaccines.
Dr Sabeti further added,
“These findings hint that current peptide pools may be incomplete.”
He and his colleagues concluded, “Therefore, unbiased approaches such as LC-MS/MS immunopeptidomics that directly identify naturally presented SARS-CoV-2 peptides on host cells can inform the design of future pools to allow more comprehensive characterization of T cell responses in COVID-19 patients.”
In simple terms, the study shows indicates that to date, we have been trying to develop therapeutics and vaccines without truly getting a full perspective first of how the various components of the immune system is responding to the SARS-CoV-2 coronavirus (antibodies, T Cells, cytokines etc) and also how the SARS-CoV-2 is also disrupting and altering a lot of these immune pathways etc. Attempts to develop and use ‘half-baked’ therapeutics and vaccines without detailed research as yet is ultimately going to backfire.!
Thailand Medical News will also be covering two more articles with detailed analysis of this study in simpler terms for common laymen to fully comprehend the implications of this study. For more on COVID-19
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