High expression of oleoyl-ACP hydrolase underpins life-threatening respiratory viral diseases.

Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah-/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease.

Structure of a fully assembled γδ T cell antigen receptor.

T cells in jawed vertebrates comprise two lineages, αß T cells and γδ T cells, defined by the antigen receptors they express-that is, αß and γδ T cell receptors (TCRs), respectively. The two lineages have different immunological roles, requiring that γδ TCRs recognize more structurally diverse ligands1. Nevertheless, the receptors use shared CD3 subunits to initiate signalling. Whereas the structural organization of αß TCRs is understood2,3, the architecture of γδ TCRs is unknown. Here, we used cryogenic electron microscopy to determine the structure of a fully assembled, MR1-reactive, human Vγ8Vδ3 TCR-CD3δγε2ζ2 complex bound by anti-CD3ε antibody Fab fragments4,5. The arrangement of CD3 subunits in γδ and αß TCRs is conserved and, although the transmembrane α-helices of the TCR-γδ and -αß subunits differ markedly in sequence, packing of the eight transmembrane-helix bundles is similar. However, in contrast to the apparently rigid αß TCR2,3,6, the γδ TCR exhibits considerable conformational heterogeneity owing to the ligand-binding TCR-γδ subunits being tethered to the CD3 subunits by their transmembrane regions only. Reducing this conformational heterogeneity by transfer of the Vγ8Vδ3 TCR variable domains to an αß TCR enhanced receptor signalling, suggesting that γδ TCR organization reflects a compromise between efficient signalling and the ability to engage structurally diverse ligands. Our findings reveal the marked structural plasticity of the TCR on evolutionary timescales, and recast it as a highly versatile receptor capable of initiating signalling as either a rigid or flexible structure.

SARS-CoV-2-specific CD8+ T cells from people with long COVID establish and maintain effector phenotype and key TCR signatures over 2 years.

Long COVID occurs in a small but important minority of patients following COVID-19, reducing quality of life and contributing to healthcare burden. Although research into underlying mechanisms is evolving, immunity is understudied. SARS-CoV-2-specific T cell responses are of key importance for viral clearance and COVID-19 recovery. However, in long COVID, the establishment and persistence of SARS-CoV-2-specific T cells are far from clear, especially beyond 12 mo postinfection and postvaccination. We defined ex vivo antigen-specific B cell and T cell responses and their T cell receptors (TCR) repertoires across 2 y postinfection in people with long COVID. Using 13 SARS-CoV-2 peptide-HLA tetramers, spanning 11 HLA allotypes, as well as spike and nucleocapsid probes, we tracked SARS-CoV-2-specific CD8+ and CD4+ T cells and B-cells in individuals from their first SARS-CoV-2 infection through primary vaccination over 24 mo. The frequencies of ORF1a- and nucleocapsid-specific T cells and B cells remained stable over 24 mo. Spike-specific CD8+ and CD4+ T cells and B cells were boosted by SARS-CoV-2 vaccination, indicating immunization, in fully recovered and people with long COVID, altered the immunodominance hierarchy of SARS-CoV-2 T cell epitopes. Meanwhile, influenza-specific CD8+ T cells were stable across 24 mo, suggesting no bystander-activation. Compared to total T cell populations, SARS-CoV-2-specific T cells were enriched for central memory phenotype, although the proportion of central memory T cells decreased following acute illness. Importantly, TCR repertoire composition was maintained throughout long COVID, including postvaccination, to 2 y postinfection. Overall, we defined ex vivo SARS-CoV-2-specific B cells and T cells to understand primary and recall responses, providing key insights into antigen-specific responses in people with long COVID.

Sequence elements within the PEXEL motif and its downstream region modulate PTEX-dependent protein export in Plasmodium falciparum.

The parasite Plasmodium falciparum causes the most severe form of malaria and to invade and replicate in red blood cells (RBCs), it exports hundreds of proteins across the encasing parasitophorous vacuole membrane (PVM) into this host cell. The exported proteins help modify the RBC to support rapid parasite growth and avoidance of the human immune system. Most exported proteins possess a conserved Plasmodium export element (PEXEL) motif with the consensus RxLxE/D/Q amino acid sequence, which acts as a proteolytic cleavage recognition site within the parasite's endoplasmic reticulum (ER). Cleavage occurs after the P1 L residue and is thought to help release the protein from the ER so it can be putatively escorted by the HSP101 chaperone to the parasitophorous vacuole space surrounding the intraerythrocytic parasite. HSP101 and its cargo are then thought to assemble with the rest of a Plasmodium translocon for exported proteins (PTEX) complex, that then recognises the xE/D/Q capped N-terminus of the exported protein and translocates it across the vacuole membrane into the RBC compartment. Here, we present evidence that supports a dual role for the PEXEL's conserved P2 ' position E/Q/D residue, first, for plasmepsin V cleavage in the ER, and second, for efficient PTEX mediated export across the PVM into the RBC. We also present evidence that the downstream 'spacer' region separating the PEXEL motif from the folded functional region of the exported protein controls cargo interaction with PTEX as well. The spacer must be of a sufficient length and permissive amino acid composition to engage the HSP101 unfoldase component of PTEX to be efficiently translocated into the RBC compartment.

Complimentary electrostatics dominate T-cell receptor binding to a psoriasis-associated peptide antigen presented by human leukocyte antigen C∗06:02.

Psoriasis is a chronic skin disease characterized by hyperproliferative epidermal lesions infiltrated by autoreactive T cells. Individuals expressing the human leukocyte antigen (HLA) C∗06:02 allele are at highest risk for developing psoriasis. An autoreactive T cell clone (termed Vα3S1/Vß13S1) isolated from psoriatic plaques is selective for HLA-C∗06:02, presenting a peptide derived from the melanocyte-specific autoantigen ADAMTSL5 (VRSRRCLRL). Here we determine the crystal structure of this psoriatic TCR-HLA-C∗06:02 ADAMTSL5 complex with a stabilized peptide. Docking of the TCR involves an extensive complementary charge network formed between negatively charged TCR residues interleaving with exposed arginine residues from the self-peptide and the HLA-C∗06:02 α1 helix. We probed these interactions through mutagenesis and activation assays. The charged interface spans the polymorphic region of the C1/C2 HLA group. Notably the peptide-binding groove of HLA-C∗06:02 appears exquisitely suited for presenting highly charged Arg-rich epitopes recognized by this acidic psoriatic TCR. Overall, we provide a structural basis for understanding the engagement of melanocyte antigen-presenting cells by a TCR implicated in psoriasis while simultaneously expanding our knowledge of how TCRs engage HLA-C.

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor.

Unlike conventional αß T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1+ and Vδ2+ γδ TCR-mediated ligand recognition, the mode of Vδ3+ TCR ligand engagement is unknown. MHC class I-related protein, MR1, presents vitamin B metabolites to αß T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2- γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3+ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3+ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Crystal structures of pertussis toxin with NAD+ and analogs provide structural insights into the mechanism of its cytosolic ADP-ribosylation activity.

Bordetella pertussis is the causative agent of whooping cough, a highly contagious respiratory disease. Pertussis toxin (PT), a major virulence factor secreted by B. pertussis, is an AB5-type protein complex topologically related to cholera toxin. The PT protein complex is internalized by host cells and follows a retrograde trafficking route to the endoplasmic reticulum, where it subsequently dissociates. The released enzymatic S1 subunit is then translocated from the endoplasmic reticulum into the cytosol and subsequently ADP-ribosylates the inhibitory alpha-subunits (Gαi) of heterotrimeric G proteins, thus promoting dysregulation of G protein-coupled receptor signaling. However, the mechanistic details of the ADP-ribosylation activity of PT are not well understood. Here, we describe crystal structures of the S1 subunit in complex with nicotinamide adenine dinucleotide (NAD+), with NAD+ hydrolysis products ADP-ribose and nicotinamide, with NAD+ analog PJ34, and with a novel NAD+ analog formed upon S1 subunit crystallization with 3-amino benzamide and NAD+, which we name benzamide amino adenine dinucleotide. These crystal structures provide unprecedented insights into pre- and post-NAD+ hydrolysis steps of the ADP-ribosyltransferase activity of PT. We propose that these data may aid in rational drug design approaches and further development of PT-specific small-molecule inhibitors.

Discovery of Anti-SARS-CoV-2 Nsp9 Binders from Natural Products by a Native Mass Spectrometry Approach.

The search for effective antiviral agents against SARS-CoV-2 remains a critical global endeavor. In this study, we focused on the viral nucleocapsid protein Nsp9, which is a key player in viral RNA replication and an attractive drug target. Employing a two-pronged approach, an in-house natural product library was screened using native mass spectrometry to identify compounds capable of binding to Nsp9. From the initial screening, apart from the previously reported hit oridonin (protein binding ratio of 0.56 in the initial screening, Kd = 7.2 ± 1.0 µM), we have identified a second Nsp9-interacting compound, the diterpenoid ryanodine, with a protein binding ratio of 0.3 and a Kd of 48.05 ± 5.03 µM. To gain deeper insights into the binding interactions and to explore potential structural requirements, the collision-induced affinity selection mass spectrometry (CIAS-MS) approach allowed us to identify six known oridonin analogues produced by the plant Rabdosia rubescens, each with varying affinities to Nsp9. Native MS validation of their individual binding activities to Nsp9 revealed that all analogues exhibited reduced affinity compared to oridonin. Structural-activity relationship analysis highlighted key functional groups, including 1-OH, 6-OH, 7-OH, and the enone moiety, which are crucial for Nsp9 binding. Combined data from our native mass spectrometry and CIAS-MS approaches provide valuable insights into the molecular interactions between Nsp9 and these compounds.

Binding of a pyrimidine RNA base-mimic to SARS-CoV-2 nonstructural protein 9.

The coronaviral nonstructural protein 9 (Nsp9) is essential for viral replication; it is the primary substrate of Nsp12's pseudokinase domain within the viral replication transcription complex, an association that also recruits other components during different stages of RNA reproduction. In the unmodified state, Nsp9 forms an obligate homodimer via an essential GxxxG protein-interaction motif, but its ssRNA-binding mechanism remains unknown. Using structural biological techniques, here we show that a base-mimicking compound identified from a small molecule fragment screen engages Nsp9 via a tetrameric Pi-Pi stacking interaction that induces the formation of a parallel trimer-of-dimers. This oligomerization mechanism allows an interchange of "latching" N-termini, the charges of which contribute to a series of electropositive channels that suggests a potential interface for viral RNA. The identified pyrrolo-pyrimidine compound may also serve as a potential starting point for the development of compounds seeking to probe Nsp9's role within SARS-CoV-2 replication.

A natural product compound inhibits coronaviral replication in vitro by binding to the conserved Nsp9 SARS-CoV-2 protein.

The Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study. Using a native mass-spectrometry-based approach to screen a natural product library for Nsp9 binders, we identified an ent-kaurane natural product, oridonin, capable of binding to purified SARS-CoV-2 Nsp9 with micromolar affinities. By determining the crystal structure of the Nsp9-oridonin complex, we showed that oridonin binds through a conserved site near Nsp9's C-terminal GxxxG-helix. In enzymatic assays, oridonin's binding to Nsp9 reduces its potential to act as substrate for Nsp12's Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. We also showed using in vitro cellular assays oridonin, while cytotoxic at higher doses has broad antiviral activity, reducing viral titer following infection with either SARS-CoV-2 or, to a lesser extent, MERS-CoV. Accordingly, these preliminary findings suggest that the oridonin molecular scaffold may have the potential to be developed into an antiviral compound to inhibit the function of Nsp9 during coronaviral replication.

Protease-associated import systems are widespread in Gram-negative bacteria.

Bacteria have evolved sophisticated uptake machineries in order to obtain the nutrients required for growth. Gram-negative plant pathogens of the genus Pectobacterium obtain iron from the protein ferredoxin, which is produced by their plant hosts. This iron-piracy is mediated by the ferredoxin uptake system (Fus), a gene cluster encoding proteins that transport ferredoxin into the bacterial cell and process it proteolytically. In this work we show that gene clusters related to the Fus are widespread in bacterial species. Through structural and biochemical characterisation of the distantly related Fus homologues YddB and PqqL from Escherichia coli, we show that these proteins are analogous to components of the Fus from Pectobacterium. The membrane protein YddB shares common structural features with the outer membrane ferredoxin transporter FusA, including a large extracellular substrate binding site. PqqL is an active protease with an analogous periplasmic localisation and iron-dependent expression to the ferredoxin processing protease FusC. Structural analysis demonstrates that PqqL and FusC share specific features that distinguish them from other members of the M16 protease family. Taken together, these data provide evidence that protease associated import systems analogous to the Fus are widespread in Gram-negative bacteria.

FusC, a member of the M16 protease family acquired by bacteria for iron piracy against plants.

Iron is essential for life. Accessing iron from the environment can be a limiting factor that determines success in a given environmental niche. For bacteria, access of chelated iron from the environment is often mediated by TonB-dependent transporters (TBDTs), which are ß-barrel proteins that form sophisticated channels in the outer membrane. Reports of iron-bearing proteins being used as a source of iron indicate specific protein import reactions across the bacterial outer membrane. The molecular mechanism by which a folded protein can be imported in this way had remained mysterious, as did the evolutionary process that could lead to such a protein import pathway. How does the bacterium evolve the specificity factors that would be required to select and import a protein encoded on another organism's genome? We describe here a model whereby the plant iron-bearing protein ferredoxin can be imported across the outer membrane of the plant pathogen Pectobacterium by means of a Brownian ratchet mechanism, thereby liberating iron into the bacterium to enable its growth in plant tissues. This import pathway is facilitated by FusC, a member of the same protein family as the mitochondrial processing peptidase (MPP). The Brownian ratchet depends on binding sites discovered in crystal structures of FusC that engage a linear segment of the plant protein ferredoxin. Sequence relationships suggest that the bacterial gene encoding FusC has previously unappreciated homologues in plants and that the protein import mechanism employed by the bacterium is an evolutionary echo of the protein import pathway in plant mitochondria and plastids.

A pocket guide on how to structure SARS-CoV-2 drugs and therapies.

The race to identify a successful treatment for COVID19 will be defined by fundamental research into the replication cycle of the SARS-CoV-2 virus. This has identified five distinct stages from which numerous vaccination and clinical trials have emerged alongside an innumerable number of drug discovery studies currently in development for disease intervention. Informing every step of the viral replication cycle has been an unprecedented 'call-to-arms' by the global structural biology community. Of the 20 main SARS-CoV-2 proteins, 13 have been resolved structurally for SARS-CoV-2 with most having a related SARS-CoV and MERS-CoV structural homologue totalling some 300 structures currently available in public repositories. Herein, we review the contribution of structural studies to our understanding of the virus and their role in structure-based development of therapeutics.

Structure-function analyses of a pertussis-like toxin from pathogenic Escherichia coli reveal a distinct mechanism of inhibition of trimeric G-proteins.

Pertussis-like toxins are secreted by several bacterial pathogens during infection. They belong to the AB5 virulence factors, which bind to glycans on host cell membranes for internalization. Host cell recognition and internalization are mediated by toxin B subunits sharing a unique pentameric ring-like assembly. Although the role of pertussis toxin in whooping cough is well-established, pertussis-like toxins produced by other bacteria are less studied, and their mechanisms of action are unclear. Here, we report that some extra-intestinal Escherichia coli pathogens (i.e. those that reside in the gut but can spread to other bodily locations) encode a pertussis-like toxin that inhibits mammalian cell growth in vitro We found that this protein, EcPlt, is related to toxins produced by both nontyphoidal and typhoidal Salmonella serovars. Pertussis-like toxins are secreted as disulfide-bonded heterohexamers in which the catalytic ADP-ribosyltransferase subunit is activated when exposed to the reducing environment in mammalian cells. We found here that the reduced EcPlt exhibits large structural rearrangements associated with its activation. We noted that inhibitory residues tethered within the NAD+-binding site by an intramolecular disulfide in the oxidized state dissociate upon the reduction and enable loop restructuring to form the nucleotide-binding site. Surprisingly, although pertussis toxin targets a cysteine residue within the α subunit of inhibitory trimeric G-proteins, we observed that activated EcPlt toxin modifies a proximal lysine/asparagine residue instead. In conclusion, our results reveal the molecular mechanism underpinning activation of pertussis-like toxins, and we also identified differences in host target specificity.

Disrupting the Allosteric Interaction between the Plasmodium falciparum cAMP-dependent Kinase and Its Regulatory Subunit.

The ubiquitous second messenger cAMP mediates signal transduction processes in the malarial parasite that regulate host erythrocyte invasion and the proliferation of merozoites. In Plasmodium falciparum, the central receptor for cAMP is the single regulatory subunit (R) of protein kinase A (PKA). To aid the development of compounds that can selectively dysregulate parasite PKA signaling, we solved the structure of the PKA regulatory subunit in complex with cAMP and a related analogue that displays antimalarial activity, (Sp)-2-Cl-cAMPS. Prior to signaling, PKA-R holds the kinase's catalytic subunit (C) in an inactive state by exerting an allosteric inhibitory effect. When two cAMP molecules bind to PKA-R, they stabilize a structural conformation that facilitates its dissociation, freeing PKA-C to phosphorylate downstream substrates such as apical membrane antigen 1. Although PKA activity was known to be necessary for erythrocytic proliferation, we show that uncontrolled induction of PKA activity using membrane-permeable agonists is equally disruptive to growth.

Antimalarial activity of novel 4-cyano-3-methylisoquinoline inhibitors against Plasmodium falciparum: design, synthesis and biological evaluation.

Central to malaria pathogenesis is the invasion of human red blood cells by Plasmodium falciparum parasites. Following each cycle of intracellular development and replication, parasites activate a cellular program to egress from their current host cell and invade a new one. The orchestration of this process critically relies upon numerous organised phospho-signaling cascades, which are mediated by a number of central kinases. Parasite kinases are emerging as novel antimalarial targets as they have diverged sufficiently from their mammalian counterparts to allow selectable therapeutic action. Parasite protein kinase A (PfPKA) is highly expressed late in the cell cycle of the parasite blood stage and has been shown to phosphorylate a critical invasion protein, Apical Membrane Antigen 1. This enzyme could therefore be a valuable drug target so we have repurposed a substituted 4-cyano-3-methylisoquinoline that has been shown to inhibit rat PKA with the goal of targeting PfPKA. We synthesised a novel series of compounds and, although many potently inhibit the growth of chloroquine sensitive and resistant strains of P. falciparum, they were found to have minimal activity against PfPKA, indicating that they likely have another target important to parasite cytokinesis and invasion.

The structure of the cytomegalovirus-encoded m04 glycoprotein, a prototypical member of the m02 family of immunoevasins.

The ability of CMVs to evade the immune system of the host is dependent on the expression of a wide array of glycoproteins, many of which interfere with natural killer cell function. In murine CMV, two large protein families mediate this immune-evasive function. Although it is established that the m145 family members mimic the structure of MHC-I molecules, the structure of the m02 family remains unknown. The most extensively studied m02 family member is m04, a glycoprotein that escorts newly assembled MHC-I molecules to the cell surface, presumably to avoid "missing self" recognition. Here we report the crystal structure of the m04 ectodomain, thereby providing insight into this large immunoevasin family. m04 adopted a ß-sandwich immunoglobulin variable (Ig-V)-like fold, despite sharing very little sequence identity with the Ig-V superfamily. In addition to the Ig-V core, m04 possesses several unique structural features that included an unusual ß-strand topology, a number of extended loops and a prominent α-helix. The m04 interior was packed by a myriad of hydrophobic residues that form distinct clusters around two conserved tryptophan residues. This hydrophobic core was well conserved throughout the m02 family, thereby indicating that murine CMV encodes a number of Ig-V-like molecules. We show that m04 binds a range of MHC-I molecules with low affinity in a peptide-independent manner. Accordingly, the structure of m04, which represents the first example of an murine CMV encoded Ig-V fold, provides a basis for understanding the structure and function of this enigmatic and large family of immunoevasins.

A structural and functional investigation of a novel protein from Mycobacterium smegmatis implicated in mycobacterial macrophage survivability.

The success of pathogenic mycobacterial species is owing in part to their ability to parasitize the generally inhospitable phagosomal environment of host macrophages, utilizing a variety of strategies to avoid their antimycobacterial capabilities and thereby enabling their survival. A recently identified gene target in Mycobacterium smegmatis, highly conserved within Mycobacterium spp. and denoted MSMEG_5817, has been found to be important for bacterial survival within host macrophages. To gain insight into its function, the crystal structure of MSMEG_5817 has been solved to 2.40 Šresolution. The structure reveals a high level of structural homology to the sterol carrier protein (SCP) family, suggesting a potential role of MSMEG_5817 in the binding and transportation of biologically relevant lipids required for bacterial survival. The lipid-binding capacity of MSMEG_5817 was confirmed by ELISA, revealing binding to a number of phospholipids with varying binding specificities compared with Homo sapiens SCP. A potential lipid-binding site was probed by alanine-scanning mutagenesis, revealing structurally relevant residues and a binding mechanism potentially differing from that of the SCPs.

CD8+ T-cell responses towards conserved influenza B virus epitopes across anatomical sites and age.

Influenza B viruses (IBVs) cause substantive morbidity and mortality, and yet immunity towards IBVs remains understudied. CD8+ T-cells provide broadly cross-reactive immunity and alleviate disease severity by recognizing conserved epitopes. Despite the IBV burden, only 18 IBV-specific T-cell epitopes restricted by 5 HLAs have been identified currently. A broader array of conserved IBV T-cell epitopes is needed to develop effective cross-reactive T-cell based IBV vaccines. Here we identify 9 highly conserved IBV CD8+ T-cell epitopes restricted to HLA-B*07:02, HLA-B*08:01 and HLA-B*35:01. Memory IBV-specific tetramer+CD8+ T-cells are present within blood and tissues. Frequencies of IBV-specific CD8+ T-cells decline with age, but maintain a central memory phenotype. HLA-B*07:02 and HLA-B*08:01-restricted NP30-38 epitope-specific T-cells have distinct T-cell receptor repertoires. We provide structural basis for the IBV HLA-B*07:02-restricted NS1196-206 (11-mer) and HLA-B*07:02-restricted NP30-38 epitope presentation. Our study increases the number of IBV CD8+ T-cell epitopes, and defines IBV-specific CD8+ T-cells at cellular and molecular levels, across tissues and age.

Mapping the immunopeptidome of seven SARS-CoV-2 antigens across common HLA haplotypes.

Most COVID-19 vaccines elicit immunity against the SARS-CoV-2 Spike protein. However, Spike protein mutations in emerging strains and immune evasion by the SARS-CoV-2 virus demonstrates the need to develop more broadly targeting vaccines. To facilitate this, we use mass spectrometry to identify immunopeptides derived from seven relatively conserved structural and non-structural SARS-CoV-2 proteins (N, E, Nsp1/4/5/8/9). We use two different B-lymphoblastoid cell lines to map Human Leukocyte Antigen (HLA) class I and class II immunopeptidomes covering some of the prevalent HLA types across the global human population. We employ DNA plasmid transfection and direct antigen delivery approaches to sample different antigens and find 248 unique HLA class I and HLA class II bound peptides with 71 derived from N, 12 from E, 28 from Nsp1, 19 from Nsp4, 73 from Nsp8 and 45 peptides derived from Nsp9. Over half of the viral peptides are unpublished. T cell reactivity tested against 56 of the detected peptides shows CD8+ and CD4+ T cell responses against several peptides from the N, E, and Nsp9 proteins. Results from this study will aid the development of next-generation COVID vaccines targeting epitopes from across a number of SARS-CoV-2 proteins.