Integrated Immunopeptidomics and Proteomics Study of SARS-CoV-2–Infected Calu-3 Cells Reveals Dynamic Changes in Allele-specific HLA Abundance and Antigen Presentation

•Host response to SARS-CoV-2 infection comprehensively deciphered.•Both class I and class II viral epitopes were identified by immunopeptidomics.•Activation of TLR3-IFN pathway and downregulation of ACE2 identified.•Allele-specific changes in HLA identified and correlates with antigen presentation. We present an integrated immunopeptidomics and proteomics study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection to comprehensively decipher the changes in host cells in response to viral infection. Immunopeptidomics analysis identified viral antigens presented by host cells through both class I and class II MHC system for recognition by the adaptive immune system. The host proteome changes were characterized by quantitative proteomics and glycoproteomics and from these data, the activation of toll-like receptor 3–interferon pathway was identified. Glycosylation analysis of human leukocyte antigen (HLA) proteins from the elution and flow-through of immunoprecipitation revealed that SARS-CoV-2 infection changed the glycosylation pattern of certain HLA alleles with different HLA alleles, showing distinct dynamic changes in relative abundance. The difference in the glycosylation and abundance of HLA alleles changed the number of strong binding antigens each allele presented, suggesting the impact of SARS-CoV-2 infection on antigen presentation is allele-specific. These results could be further exploited to explain the imbalanced response from innate and adaptive immune system in coronavirus disease 2019 cases, which would be helpful for the development of therapeutics and vaccine for coronavirus disease 2019 and preparation for future pandemic. We present an integrated immunopeptidomics and proteomics study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection to comprehensively decipher the changes in host cells in response to viral infection. Immunopeptidomics analysis identified viral antigens presented by host cells through both class I and class II MHC system for recognition by the adaptive immune system. The host proteome changes were characterized by quantitative proteomics and glycoproteomics and from these data, the activation of toll-like receptor 3–interferon pathway was identified. Glycosylation analysis of human leukocyte antigen (HLA) proteins from the elution and flow-through of immunoprecipitation revealed that SARS-CoV-2 infection changed the glycosylation pattern of certain HLA alleles with different HLA alleles, showing distinct dynamic changes in relative abundance. The difference in the glycosylation and abundance of HLA alleles changed the number of strong binding antigens each allele presented, suggesting the impact of SARS-CoV-2 infection on antigen presentation is allele-specific. These results could be further exploited to explain the imbalanced response from innate and adaptive immune system in coronavirus disease 2019 cases, which would be helpful for the development of therapeutics and vaccine for coronavirus disease 2019 and preparation for future pandemic. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which was declared a pandemic in March 2020. Since then, the world has witnessed the mobilization of the global scientific community toward the fastest vaccine development in history. Currently, there are 14 vaccines approved by WHO for emergency use globally, with another 341 in various stages of development. Three years after the discovery of the novel coronavirus, there is still a need to understand how the virus mobilizes the host cellular pathways to allow rapid infection and replication and identify the key viral epitopes that stimulate the host immune system to generate protective immunity. SARS-CoV-2 is a beta coronavirus, a member of the Coronaviridae family of viruses, which include four subgroups, denoted alpha, beta, gamma, and delta. Together, the family infect a wide range of mammal and bird species, with mutations allowing the viruses to cross the species barrier. SARS-CoV-2 has a 30 kb positive–sense RNA genome, with 14 ORFs that encode a total of 29 proteins. These include four structural proteins (spike, envelope, membrane, and nucleocapsid protein), 16 nonstructural proteins (Nsp 1–16) and nine accessory proteins (Orf 3a, Orf 3b, Orf6, Orf 7a, Orf 7b, Orf8, Orf 9b, Orf 9c, and Orf 10) (1Yao H. Song Y. Chen Y. Wu N. Xu J. Sun C. et al.Molecular architecture of the SARS-CoV-2 virus.Cell. 2020; 183: 730-738Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar). Built on the knowledge of related coronaviruses, recent studies have illuminated the life cycle of SARS-CoV-2 (2Chu H. Chan J.F.-W. Yuen T.T.-T. Shuai H. Yuan S. Wang Y. et al.Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study.Lancet Microbe. 2020; 1: e14-e23Abstract Full Text Full Text PDF PubMed Google Scholar). This virus enters cells via the binding of the spike structural protein to human angiotensin–converting enzyme 2 (ACE2) on the surface of host cells. Intracellularly, the viral genome is transcribed by the host ribosomes. The viral genome is replicated by the viral RNA–dependent RNA polymerase, on membrane structures derived from the endoplasmic reticulum (ER). Assembly of mature virions occurs in the ER-Golgi intermediate compartment, budding from the ER-Golgi membranes before been released outside the cells via an exocytosis like process (3Fung T.S. Liu D.X. Human coronavirus: host-pathogen interaction.Annu. Rev. Microbiol. 2019; 73: 529-557Crossref PubMed Scopus (652) Google Scholar). Despite the rapid progress made by the scientific community, the emergence of variants of concern (VoCs) continues to challenge vaccine and drug developers. Although the currently approved vaccines showed excellent efficacy toward the original SARS-CoV-2, vaccine breakthrough infections have been observed with several VoCs. This continues to highlight the need to build on our current knowledge of the host response to the virus, in particular the modified signaling pathways to allow viral infection and viral epitopes presented to the immune system. The genome sequence of SARS-CoV-2 was widely disseminated in the early 2020 (4Wu S. Chen Wang Song et coronavirus with human respiratory disease in 2020; PubMed Scopus Google the rapid of with the novel coronavirus both and Although vaccines the original virus and in of of infection and of there is still a need for vaccines and vaccines that from current and variants of are viral to an need to understand immune to viral for how cells could variants H. cells could in of PubMed Scopus Google Scholar). infect host cells and proteins are and presented on the surface of human cells by cells antigens and an immune response that the cells. The of the presented on MHC is the and knowledge of the on that cells. 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