Marine natural products occurring along the Western Australian coastline are the focus of this review. Western Australia covers one-third of the Australian coast, from tropical waters in the far north of the state to cooler temperate and Antarctic waters in the south. Over 40 years of research has resulted in the identification of a number of different types of secondary metabolites including terpenoids, alkaloids, polyketides, fatty acid derivatives, peptides and arsenic-containing natural products. Many of these compounds have been reported to display a variety of bioactivities. A description of the compound classes and their associated bioactivities from marine organisms found along the Western Australian coastline is presented. Natural products have long played an important role both as direct agents and as molecular scaffolds providing inspiration for novel pharmaceuticals . NotablyHistorically, marine natural product research has lagged behind its terrestrial counterpart due to the inaccessibility of samples, as well as the absence of ethnobotanical knowledge in guiding the selection of taxa for investigation . The devWestern Australia covers one-third of Australia’s coast, from tropical waters in the far north of the state to cooler temperate and Antarctic waters in the state’s south . The sta150, 151) from the marine sponge Phorbas sp. . . Spongia14–16 were reported from a collection of Higginsia sp.  collected off Lancelin. The structure of 14 was verified via single-crystal X-ray diffraction [16. Subsequent reinvestigation of the non-polar fractions of the lipophilic sponge extract afforded the tricyclic diterpene hydrocarbon 17 and the daucadiene sesquiterpene 18  collected from Exmouth gulf afforded the novel linear furanoditerpenes 12-hydroxy ambliofuran (19) and 12-acetoxyambliofuran (20)   18]. Mo. MoSpong7 and 28  [18].2Cl2 soluble fractions of a Hippospongia sp.  collected from south of the Great Australian Bight afforded six new C-25 derived linear furanoterpenes, given the trivial names hippospongins A–F (29–34) (29–34. Hippospongin A (29) exhibited mild antibiotic activity, inhibiting the growth of Staphylococcus aureus at concentrations of circa 200 µg/disk in a standard agar plate assay [The CH (29–34)  19]. Th. Th2Cl2 Clathria sp.  collected off the Great Australian Bight yielded the novel compounds clathrins A, B and C (35–37) (35) is postulated to provide support for the biosynthetic origins of other marine meroterpenoids derived via a mixed shikimate-terpenoid biosynthetic pathway. Attempts to elucidate the stereochemistry of clathrin B (36) were thwarted by the facile atmospheric oxidation of 36 to compound 37; furthermore, the absolute configuration of 35 remains unresolved , is, isDarwi4 (52) and A5 (53)  collected near Scott Reef, along with five known sarasinosides [52 is unusual in the composition of its bis-enol ether moiety [Sarasinosides A A5 (53)  were isoinosides . The comr moiety . Stelletta sp.  collected by trawling operations in the Great Australian Bight was the source of the terpenyl-pyrrolizidine conjugates bistelletazines A–C (54–56) and the cyclic terpenyl-imidazole conjugate macrocycle bistelletazole A (57) (A sample of e A (57)  27]. Th. ThStell58), 19-aminoarenarone (59), 18-methylaminoarenarone (60) and 19-methylaminoarenarone (61), and the new dimeric popolohuanone F (62) were isolated from a Dysidea sp.  collected from Scott Reef . Fu. Fu65), Iotrochota sp.  collected from the Five Fathom Bank, off the coast of Fremantle, was reported to yield the novel metabolite (E)-3-(6-bromoindol-3-yl)prop-2-enoate (73)   30]. Th. ThIotro74), bastadin 20 (75) and the sulfate half-esters 15,34-O-bis-sulfatobastadin 7 (76) and 10-O-sulfatobastadin 3 (77)  collected from Stuarts Shoal, Exmouth Gulf . Th. ThCymbaEchinodictyum sp.  collected in the Great Australian Bight afforded four novel compounds, echinosulfone A (81a) and the echinosulfonic acids A–C (82a–84a) (81b–84b (85) [86) (Haemonchus contortus with echinobetaine B (86) exhibiting an LD99 of 8.3 µg/mL. The structures of racemic echinobetaines A and B have also been confirmed via total synthesis [The ethanolic extracts of an 82a–84a)  33]. Th. ThEchin  and (+)-85) [86)  38] as  as EchinZyzzya sp.  collected at Assail Bank, between North Island and the Wallab Group, and a Latrunculia purpurea  collected on Horse Shoe Reef, west-northwest of Margaret Brock Lighthouse, were both reported to yield the novel pyrolloiminoquinone pigment discorhabdin Q (87)  in the NCI 60 cancer cell line panel [A n Q (87)  39]. Th. ThZyzzyJaspis sp.  collected off a low, uninhabited rocky island near the northwestern end of Serrurier Island, afforded the novel alkaloids bengamides Y (88) and Z (89) and bengazole Z (90) (91) and B (92). Biological investigation revealed that metabolites 88 and 89 exhibited specific cytotoxicity against 10 cancer cell lines . In. InJaspine) (97) . The rel studies .Clathria sp.  collected by trawl off the coast of Cape Arid yielded the new tricyclic guanidine alkaloid mirabilin G (98) (99) and F (100) and the novel mirabilins H, I and J (101–103) . Re. ReClath101–103) . Mirabilrst time . The aut M (105)  44]. Mi. MiClathXestospongia sp.  collected at Benetts Shoal, Exmouth Gulf, afforded the dimeric 2,9-disubstituted-1-oxaquinolizidine alkaloids (+)-xestospongin A (106), (−)-xestospongin C (107) and (+)-xestospongin D (108), as well as arugospongine C (109), (+)-7S-hydroxyxestospongin A (110) and (+)-demethylxestospongin B (111) (S-hydroxyxestospongin A (110) was solved using single-crystal X-ray diffraction, and the absolute configuration was secured using Mosher’s ester analysis. The absolute configuration of (+)-xestospongin D (108) was secured by analysis of anomalous dispersion in single-crystal X-ray diffraction experiments [106–109 and 111 exhibited modest antifungal activity (MIC 30–100 μg/mL) against various fluconazole-resistant Candida sp. [The methanolic extracts of a  B (111)  45]. Th. ThXesto112) and batzelline D (113), along with the known pigments batzelline C (114), isobatzelline C (115) and makaluvamine D (116)  collected off Abrolohos Island . Th. ThAxine129–132) . The pla reports . Further-workers . Six new134–138) , have beuth Gulf . The relf Me-C-8 .139) and B (140), as well as an artefact of extraction (141) and the enantiomer of a known compound (148), were isolated from a Pseudoceratina cf. verrucosa  collected off the Dampier Peninsula . Th. ThPhorb154–156) , the hig) . Th. ThPhorbRaspailia  sp.  collected by trawl on the northern Rottnest Shelf afforded the known compounds phorboxazoles A and B  (163), isolated for the first time as a natural product . Th. ThAmphiGeodia sp.  collected in the Great Australian Bight yielded a new macrocyclic polyketide lactam tetramic acid, as a magnesium salt 170 (170) was determined spectroscopically. The magnesium content of the sample was determined by energy-dispersive spectroscopy and atomic absorption spectroscopy allowing the authors to deduce the presence of one unit of magnesium for every two units of tetramic acid . Th. ThOceanLC-ESIMS . The strOceanapia sp. collected by dredge off Scott Reef afforded the hybrid α,ω-bifunctionalised sphingoid tetrahydroisoquinoline β-glycoside oceanalin A (183), as well as the known compound rhizochalin (184)  were isolated from the same Oceanapia sp. collected off Scott Reef [Oceanapia sp. was isolated a ceramide fraction characterised by methanolysis and GC-MS analysis [Oceania sp. afforded the new bolaampiphilic sphingoid bases rhizochalin B (187) and rhizochalinin B (188)   71]. Th. ThOceanott Reef . The cerott Reef . Also franalysis . Most re B (188)  characteMycale sp.  collected by benthic sled off the coast of Albany afforded seven novel polyacetylene nitriles: albanitriles A–G (189–195) (191 and 195 were isolated as racemic mixtures. Albanitrile C (191) exhibited mild toxicity towards Bacillus subtilis at 90 μM. Additional investigations revealed that compounds 189–192 exhibited activity against Giardia duodenalis, with albanitrile A (189) exhibiting activity at a minimum concentration of 12 μM, which was comparable to metronidazole used as the positive control. Compounds 189 to 191 also exhibited weak inhibition against Tritrichomonas fetus, on the order of 200 μM [A 189–195)  75]. Th. ThMycalTheonella sp.  collected by SCUBA near Perth, off Cape Vlamingh, afforded the cyclic octapeptide perthamide B (196)   76]. Th. ThTheon197, 198)  collected in the states northwest pyrimidine (199)   for the odilator . 200), previously reported by Bergmann et al. from Cryptotethya crypta and 2′-deoxyspongosine (201)  , previouHaliclona sp.  collected from Cosy Corner off the southwest coast of Western Australia . Th. ThTrachErylus sp. , collected west of Margaret River, yielded the first naturally occurring cyclo-nucleoside N-3,5′-cycloxanthosine (207) (An ne (207)  84]. Th. ThEryluHaliclona sp.  collected from Exmouth Gulf afforded 1,4-dideoxy-1,4-imino-D-arabinitol (207)  as well as 1,4-dideoxy1,4-imino-D-xylitol (209), present both in a Haliclona sp. and Raspalia sp. Investigation of a Cymbastela sp.  revealed the presence of two additional isomers, identified tentatively as the diastereomers 1,4-dideoxy-1,4-imino-D-ribitol (210) and 1,4-dideoxy-1,4-imino-D-arabitol (211)  , for theol (211)  by Marfe and 211 . Compounactivity .212) and 3,5-dibromo-2-phenol (213) phenol (ol (213) , were isfraction .Dysidea dendyi  collected by hand from Scott Reef at a depth of 3 m afforded two new tetrabromodibenzo-p-dioxins: spongiadioxins A and B   and its methyl ether 217 as well as the related diphenyl ethers 218–220 . Th. ThDysid 218–220 . The strectively .Ctenocella pectinata , collected in Exmouth Bay, afforded three new sterols, pectinoacetals A–C (221–223) (A sample of 221–223) , isolateEleuthorobia sp. , found near Bennett’s Shoal, yielded the new diterpene glycoside eleutherobin (224)  exhibited significant specific cytotoxicity against a diverse panel of breast, renal, ovarian and lung cancer cell lines with an IC50 range of 10–15 nM. Compound 224 was found to stabilize microtubules by competing for the paclitaxel binding site on the microtubule polymer [Bioassay-guided fractionation of a rare Alcyonacean soft coral in (224)  10]. Th. ThEleutBriareum excavatum , collected at Rowley Shoals, afforded the known diterpene Z,10R*,11R*,12S*,14S*)-2,3,14-triacetoxy-11,12-epoxybriara-5,8(17)-dien-18-one (225), as well as the new briarane diterpenes excavatolides N–T (226–232)  by geometry optimisation of the proposed structure using density functional theory. Compounds 226–229 exhibited various levels of cytotoxic activity against P388 murine leukaemia, A549 human lung carcinoma, HT29 human colon carcinoma and MEL28 human melanoma cells [Fractionation of a freeze-dried specimen of 226–232)  90]. Th. ThBriarAnthoptilum cf. kukenthali , collected by dredge at a depth of 267 m, northwest of Port Hedland, afforded five new briarane diterpenoids: the anthoptilides A–E (233–237) (233) was solved by single-crystal X-ray diffraction. Compounds 223–237 inhibited [3H]CPDPX binding to rat brain adenosine A1 receptors with IC50 values of 420 µM, 45 µM, 3.1 µM, 500 µM and 490 µM, respectively . Th. ThMacro methods . Compounne panel . Additio245 , collected from the Montebello Archipelago, afforded three known cyclic peptides, ulithiacyclamide (246), lissoclinamide (247) and patellamide B (248), as well as the novel patellamide F (249) (249) was confirmed by Marfey’s analysis. Patellamide B (248), patellamide F (249) and ulithiacyclamide (246) exhibited modest general cytotoxicity in the NCI 60 cell line human tumour screen with LC50 values of 48 µM, 13 µM and 3 µM, respectively [Bioassay-guided fractionation of the colonial ascidian  F (249)  96]. Th. ThLisso250)  -4-methylpentan-2-one (255) -4,8-dimethylnonan-4-olide [Fresh specimens of a tunicate tentatively identified as ne (255)  98]. Th. ThAplidDidemnum sp. , collected by hand using SCUBA, near Exmouth, afforded the known bacterial metabolite enterocin (256) as well as 5-deoxyenterocin (257), previously reported in a Japanese patent albeit with no spectroscopic data reported. Reversed-phase HPLC of the lipohilic fractions afforded the novel esters enterocin-5-behenate (258) and entorocin-5-arachidate (259)  , assigneDidemnum sp. , collected near Ningaloo Reef, yielded four new aromatic alkaloids ningalins A–D (260–263)  , assigneanalysis . The autcaffolds .Aplidiopsis sp.  collected near Ningaloo Reef afforded the zwitterionic hydroxyadenine aplydiamine (264)  . The str protons . 265 and 266)  pyran-4-one (277)  afforded the known compounds rhodolamprometrin (278) and rhodocomatulin-6,8-dimethyl ether (279). Chemical investigation of Comatula rotolaria afforded compounds 278 and 279 as well as rhodocomatulin-6-methyl ether (280) , t, tEncyotCystophora harveyi , collected by SCUBA from the first bay to the east of the Cape Leeuwin lighthouse, yielded the new linearly fused tricyclic compound pycnanthaquinone C (295), the structure of which was elucidated spectroscopically . T. TLaurenynthesis . Vidalia spiralis  collected at Yanchep yielded the new halogenated diol 3,4-dibromo-5-methylenecyclopent-3-ene-1,2-diol (300)   as a fincompound . Laurencia filiformis  yielded the novel brominated eudesmane sesquiterpenes austradiol acetatete (301) and austradiol diacetate (302) as well as the known cis-dihydrorhodophytin (303) and cis-epidihydrorhodophytin (304)  was provided by complexation with a europium chemical shift reagent [Fractionation of the lipophilic extracts of a sample of the red algae in (304)  116]. T. TLaurenHypnea valendiae , collected at Quobba Lagoon, returned 4-amino-7-(5′-deoxyribose-1′β-yl)-5-iodopyrrolo pyrimidine (305)   as the plidation .Plocamium mertensii , collected at Carnac Island, yielded -2-bromo-l-chloro-4-(2′-chloroetheny1)-1-methyl-5-methylenecyclohexane (306). The structure of 306 was solved by single-crystal X-ray diffraction ..LaurenciCaulerpa flexilis var. muelleri  collected from Cosy Corner. The structure of the metabolites was confirmed as -2-[2′-ethyl]buta-1,3-diene-I,4-diyl diacetate (314) and (2E)-3-formyl-5-pent-2-enyl acetate (315)   using cosignment .Caulerpa trifaria  collected at Point Peron afforded the new sesquiterpene metabolite 316, the structure of which was deduced spectroscopically. The absolute configuration of the compound remains unknown. Samples of C. brownii, C. pexilis, C. peltata and C. racemosa also collected from Point Peron failed to yield 316. However C. peltata and C. racemosa afforded caulerpin (317) in low yield as red-plate crystals . T. TAphano320)  and Prorocentrum minimum . T. TAspergAspergillus noonimiae collected in waters near Perth afforded the indolic diterpenes noonindoles A–F (331–336)   as well albicans .Panulirus cygnus (George) , afforded arsenobetaine (337)   as the pynthesis .Ecklonia radiata  afforded the metabolites 2-hydroxy-3-sulphopropyl-S-deoxy-5-(dimethylarsenoso)furanoside (338) and a 2,3-dihydroxypropyl-S-deoxy-S-(dimethylarsenoso)furanoside (339)  [Ecklonia radiata yielded the minor metabolite 3-glycerophosphoryl-2-hydroxy-I-[5-deoxy-5-(dimethylarsinoyl)-β-ribofuranosyloxy]propane (341). The structure of this metabolite was elucidated principally by NMR spectroscopy [Investigation of the arsenical constituents of the brown kelp de (339)  126]. T. TEcklonol (340) . Additiotroscopy .342, as well as (2S)-3-[5-deoxy-5-(dimethylarsinoyl)-β-D-ribofuranosyloxy]-2-hydroxypropyl hydrogen sulfate (343), was isolated from the kidney of the giant clam, Tridacna maxima , collected from Shark Bay [343 was solved using single-crystal X-ray diffraction. Targeted isolation of arsenical metabolites from the brown alga Sargassum lacerifolium  afforded two new ribosides methyl 5-deoxy-5-(dimethylarsinoyl)-β-D-riboside (344) and 1-O-[5′-deoxy-5′-(dimethylarsinoyl)-β-D-ribosyl] mannitol (345), in addition to five known arsenic-containing ribosides taurine (350). The second collection also afforded the previously reported (2S)-3-[(5′-deoxy-5′-trimethylarsonio-β-D-ribosyloxy)-2-hydroxypropyl] sulfate (351). The structures of 346 to 351 were validated by total synthesis [346 to 351 deriving from donation of all three alkyl groups present on S-adenosyl methionine to inorganic arsenate present in seawater [Two batches of eviously  131]. T. TTridacseawater .Here we have reviewed the marine natural products that have been reported from the fauna and flora of Western Australian waters. This review describes the identification of over 350 metabolites representing a diverse array of chemical compounds that have been reported over the past 40 years. Most of the compounds have also been reported to display some biological activity in line with the high rates of bioactivity studies of marine natural products reported elsewhere ,133.A statistical analysis of the distribution of marine natural products from various taxonomic sources and the percentage of each structural class among all the marine natural products reported from Western Australia  reveals While there has been extensive research into marine natural products originating from other major marine biodiversity hotspots, such as the Americas, Southeast Asia, Japan, Eastern and Southern Australia and New Zealand, there have been relatively few major studies of marine natural products from Western Australia. This is the largest coastline of Australia, and biodiversity studies suggest that Western Australia marine areas are a source of significant biodiversity with many of the species remaining largely uncharacterised and underexplored ,136,137.In an age of superbugs and viral pandemics, the need for discovering new anti-infective agents is paramount ,139, and


Our data revealed a substantial number of SARS-CoV-2-specific T cells that expressed memory phenotype markers. Integrated phenotypic analyses demonstrated diverse pre-existing memory states that included cells with distinct polarization states and trafficking potential to barrier tissues. T cell clones generated from tetramer-labeled cells cross-reacted with antigens from commensal bacteria in the skin and gastrointestinal tract. Direct ex vivo tetramer staining for one spike-specific population showed a similar level of cross-reactivity to sequences from endemic coronavirus and commensal bacteria. These data highlight the complexity of precursor T cell repertoire and implicate non-infectious exposures to common microbes as a key factor that shapes human pre-existing immunity to SARS-CoV-2.The baseline composition of T cells directly impacts later response to pathogens, but the complexity of precursor states remains poorly defined. Here, we examined the baseline state of SARS-CoV-2-specific T cells in unexposed individuals. SARS-CoV-2-specific CD4 Pre-existing immunity to SARS-CoV-2 displayed diverse phenotypes, had broad tissue tropism and cross-reacted with commensal antigens. SARS-CoV-2 infection (COVID-19) presents with a myriad of organ involvement, variable duration of symptoms, and diverse clinical presentations that range from asymptotic infection to death 1–6. TCoronaviridae family than expected by chance  tetramer enrichment. Blood samples collected prior to year 2020 were used to ensure the absence of prior SARS-CoV-2 exposure. The activation of naïve T cells in an anatomical niche imprints selective trafficking properties . A set of 12 peptides that stimulated T cells in COVID-19 patients based on prior studies and predicted to bind HLA-DR4 were selected for generation of tetramers (Table S2). We coupled tetramer staining with magnetic column-based enrichment to enable enumeration of rare tetramer-labeled T cells in the unprimed repertoire. The baseline differentiation states of tetramer-labeled T cells were delineated by anti-CD45RO and CCR7 antibody staining. In total, we analyzed 117 SARS-CoV-2-specific CD4+ populations in 12 healthy unexposed adults. SARS-CoV-2 precursors varied by epitope-specificity and showed heterogeneity between individuals  using pre-pandemic blood samples collected prior to 2020 (Table S1). SARS-CoV-2-specific CD4ividuals . Next, wcination 30. Becacination , Fig. S2+ cells, suggesting that some pre-existing cells perceived stimulation signals at baseline  T cells (CXCR5) or Th1 cells (CXCR3), suggesting polarization of some pre-existing memory cells into defined T helper subsets . Memory phenotype clusters were further separated by gut and skin trafficking receptors, which localized to distinct parts of the UMAP and co-stained with chemokine receptors in distinct patterns . By contrast, S236-labeled T cells differed from other SARS-CoV-2-specific populations and primarily mapped to cluster 6 on the UMAP  or without (CD25lo) Treg features. CD25lo cells were further gated by CXCR5 and CXCR3 staining to mark Tfh and Th1-associated phenotypic subsets . We did not focus on Tregs due to low frequency of CD25hiCD127lo cells in both HA- and SARS-CoV-2 tetramer-labeled populations. For CXCR3 and CXCR5, SARS-CoV-2-specific T cells contained a lower proportion of CXCR3+CXCR5+ and CXCR3+CXCR5- cells, but the percentage of CXCR3-CXCR5+ subset was not significantly different between SARS-CoV-2 precursors and HA-specific T cells . Expanded T cells were re-stained by tetramers and tested for response to S936 peptide. In total, we generated 17 clones and confirmed the correct specificity for 16 clones by tetramer staining and/or peptide response . Six candidate peptides from commensal bacteria identified based on similarity to the predicted S936 core sequence were used to investigate the ability of S936-specific T cells to respond to microbe-derived sequences (Table S3). We tested twelve S936 clones that grew well in culture for TNF-α production after a 5-hours stimulation with peptide-treated monocyte-derived DCs. This revealed high levels of responses to non-spike sequences. S936 clones were most responsive to P3, P5, and P6, and had cross-reactivity to at least one microbial peptide per clone . In total, 25 SARS-CoV-2-specific T cell clones were stimulated with five cultured bacterial lysates. This identified two responsive clones from different donors that recognized distinct SARS-CoV-2 epitopes  compared to phosphate buffered saline (PBS) . Pre-treepitopes . The S93P. copri . The 2B8. ovatus . These d+ T cells that bound P3 and P5 tetramers but not HKU1 or OC43 tetramers by direct ex vivo staining  for normalization (HIS-tagged HLA-DR protein monomers with tethered thrombin cleavable CLIP peptide were produced by Hi5 insect cells and extracted from culture supernatant using Ni-NTA (Qiagen). HLA-DR monomers were biotinylated overnight at 4°C using BirA (Avidity), analyzed by streptavidin gel-shift assay to confirm biotinylation, and purified by size exclusion chromatography using Superdex 200 size exclusion column . Peptide exchange and tetramerization were performed using standard protocols as previously described 28, 59Prevotella copri DSMZ 18205, Staphylococcus epidermidis ATCC 14990, Akkermansia muciniphila ATCC BAA-835, Porphyromonas gingivalis ATCC 33278, and Bacteroides ovatus ATCC 8483 were obtained from DSMZ  or ATCC  and grown in Schaedler Broth, Nutrient Broth, Brain Heart Infusion Broth, Supplemented Tryptic Soy Broth, or Modified Chopped Meat Medium, respectively. P. copri, A. muciniphila, and B. ovatus were grown in an anaerobic glove box containing approximately 2.5% CO2, 2.5% H2, and 95% N2. All strains were grown at 37°C until the mid- to late-log phase growth. 250 ml of the culture was spun down at 3000 g for 10 min, the supernatant was removed, the pellet was resuspended in 4 ml of PBS, and sonicated using a probe sonicator  for the preparation of bacterial lysates. The bacterial lysates were clarified by centrifugation at 10,000 rpm for 1 min at 4°C. The clarified bacterial lysates were then treated with Polymyxin beads (Sigma) to remove endotoxin content followed by estimation of total protein content in the lysates through BCA protein assay method (ThermoFisher). Fecal lysates were generated by diluting 100 mg of fecal sample in 1 ml PBS and sonicated 3 times for 1 min at 4°C with 30 s of rest in between. The fecal lysates were clarified by centrifugation at 15000 rpm for 5 min before use.Generation of T cell clones: Cells were stained with tetramers and enriched by magnetic isolation as described above. Tetramer-positive cells were sorted as single cells into 96-well plates using the purity mode on FACSAria II (BD). Each well contained 105 irradiated PBMCs and 104 JY cell line (an Epstein–Barr virus (EBV)-immortalized B cell lymphoblastoid line, ThermoFisher), PHA , IL-7 , and IL-15 . IL-2  was added on day 5 and replenished every 3-5 days. Cells were resupplied with fresh medium with IL-2 (50 IU/ml), PHA (1:100) and 105 irradiated PBMCs every two weeks.Generation of DCs: Monocytes from PBMCs of HD1 and HD2 were isolated using RosetteSep Human Monocyte Enrichment Cocktail (StemCell). 5 million cryopreserved monocytes were thawed in 6.4 ml of DCs media  containing 100 ng/ml GM-CSF and 500 U/ml IL-4. On the third day, 3.2 ml fresh media with 100 ng/ml GM-CSF, 500 U/ml IL-4, and 0.05 mM 2-mercaptoethanol were added to the ongoing culture. For maturation of DCs with bacterial lysates or fecal lysates, the immature DCs were harvested on day 4 after thawing. 25,000 DCs were plated in U bottom 96-well plate, incubated with 50 ug/ml polymyxin bead treated bacterial lysates or 50 ug/ml fecal lysates for 36h, followed by the addition of LPS (100 ng/ml) on day 6. The bacterial lysates or fecal lysates treated DCs were co-cultured with T cell clones on day 7. For peptide stimulation, immature DCs were harvested on day 5 after thawing. 0.1 million DCs were plated in a flat bottom 96-well plate and matured with LPS (100 ng/ml) and incubated with peptide (20 ug/ml) for 16-24h before co-culturing with T cells.Stimulation of T cell clones: T cell clones were rested overnight in fresh media without IL-2 and added to wells containing matured DCs. Cells were incubated for 5 hours in the presence of monensin  and Brefeldin A . For MHC blocking experiments, anti-MHC class II antibodies, L243  and TU39 , were added to T cells before combining with DCs. Intracellular cytokine staining with anti-TNF-α and anti-IFN-γ (BioLegend) was performed after stimulation using BD Cytofix/Cytoperm Fixation/Permeabilization Kit according to manufacturer protocol (BD).Bacterial peptides: To identify potential cross-reactivity to microbial peptides, we predicted the DR4 binding register in S936 using NetMHCII 2.3. We then performed a Protein BLAST analyses against the non-redundant protein sequence database using the S936 core sequence (LSSTASALG), excluding SARS-CoV-2 and coronavirus related sequences . We curated six sequences from commensal bacteria allowing for 1 amino acid mismatch and extended these 9-mer peptides by two amino acids on both ends based on the bacterial protein sequence.Coronavirus sequences: All spike and non-spike sequence of SARS-CoV-2 and common coronaviruses were derived from NCBI database. . Pair-wise alignment using Clustal Omega was performed to identify aligned regions. Similarity between the aligned sequences were calculated by Sequence Identity And Similarity (SIAS) using BLOSUM62 matrix. A score of 0 was used if no aligned region was found. Missing sequences were excluded from the analyses.+ cells stained using pooled or individual tetramers were exported from Flowjo. A total of 932 tetramer+ cells were read into R by flowCore and combined into one single dataset for subsequent data processing and high-dimensional analyses using the Spectre package in R , Friedman’s repeated measures one-way ANOVA (Dunn), or two-way ANOVA (Tukey) was significant. Statistical analyses were performed using GraphPad Prism. Lines and bars represent mean and variability is represented by standard error of the mean (SEM). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns (not significant).Data transformation was performed using the logarithmic function or inverse hyperbolic sine transformation (


This tumor-suppressive cell competition restrains EGFR and Hippo signaling and enables Eiger-JNK mediated apoptosis in scrib−/− clones. Here, we show that the activation state of the endosomal actin regulator WASH is a central node linking EGFR and Hippo signaling activation. The tyrosine kinase Btk29A and its substrate WASH are required downstream of Ptp10D for “loser” cell elimination. Constitutively active, phosphomimetic WASH is sufficient to induce both EGFR and Yki activation leading to overgrowth. On the mechanistic level we show that Ptp10D is recycled by the WASH/retromer complex, while EGFR is recycled by the WASH/retriever complex. Constitutive WASH activation selectively interferes with retromer function leading to Ptp10D mistargeting while promoting EGFR recycling and signaling activation. Phospho-WASH also activates aberrant Arp2/3 actin polymerization, leading to cytoskeletal imbalance, Yki activation and reduced apoptosis. Selective manipulation of WASH phosphorylation on sorting endosomes may restrict epithelial tumorous growth.Cell competition is a conserved homeostatic mechanism whereby epithelial cells eliminate neighbors with lower fitness. Cell communication at the interface of wild-type “winner” cells and polarity-deficient ( Drosophila and in proliferating epithelial cells, WASH activation differentially affects the separate recycling routes of EGFR and its inhibitor Ptp10D.Polarity-deficient epithelial cells are eliminated through Sas-Ptp10D-mediated suppression of EGFR signalling during cell competition. Here the authors show the constitutively active, phosphomimetic WASH can activate both EGFR and Hippo signalling and thereby, induces the overgrowth of these polarity-deficient cells in  Drosophila and mammals2. To maintain tissue homeostasis, epithelial cells communicate with their neighbors and remove unfit or mutant cells with oncogenic mutations. Cell competition can be induced by competing for survival factors and nutrients11, fitness sensing15 or mechanical force16. For instance, mutations in the tumor suppressor gene scribble (scrib), involved in epithelial polarization and endocytosis, generate imaginal disc overgrowth. However, when surrounded by wild-type neighbors, scrib−/− cells are eliminated through Eiger activation of JNK-mediated apoptosis19. In eye-antennal imaginal discs, elimination initiates from translocation of the receptor tyrosine phosphatase Ptp10D to the lateral membranes of scrib−/− cells, where it associates with its ligand Sas at the adjacent membranes of wild-type cells. This enables Ptp10D to suppress EGFR signaling in scrib−/− leading to their elimination through apoptosis14. The underlying mechanism of how interface communication between tumor and normal cells is maintained and how it controls signaling in scrib−/− clones remains unclear. WASH (Wiskott–Aldrich syndrome protein and SCAR homologue), controls endosomal fission and cargo sorting by facilitating F-actin polymerization via the Arp2/3 complex22. Mammalian and Drosophila WASH associate with SWIP, Strumpellin, FAM21 and CCDC53 to form the WASH regulatory complex (SHRC)25. WASH is required for retromer function in mammalian cell lines21 and for selective retrograde recycling in Drosophila26. In the Drosophila embryo, the Ptp10D/Btk29A/WASH circuit controls the initiation of an apical endocytosis burst leading to luminal protein clearance and airway maturation. Btk29A phosphorylates and activates WASH, while the receptor tyrosine phosphatase Ptp10D antagonizes WASH phosphorylation. This mode of WASH activation by phosphorylation also operates in mouse fibroblasts27.Cell competition during epithelial development is a common mechanism operating in scrib−/−-induced tumor-suppressive cell competition. A phosphomimetic WASH construct is sufficient to induce over-proliferation in scrib−/− clones by activating EGFR signaling and the Hippo effector yki. Our work proposes a bifurcate recycling mechanism for EGFR and its inhibitor Ptp10D in epithelial cells during tumor-suppressive cell competition. Phospho-WASH is differentially regulated in the distinct recycling routes, offering possibilities to restrain aberrant growth signaling from EGFR.In this work, we show that WASH functions downstream of Ptp10D/Btk29A in the scrib mutant clones are often eliminated from the eye disc epithelium, when surrounded by wild-type cells whereas scrib−/− Ptp10DRNAi clones in the same settings show overgrowth14  in the scrib−/− Ptp10DRNAi clones. Dampening Btk29A activity strongly suppressed scrib−/− Ptp10DRNAi clone overgrowth, suggesting that Btk29A antagonizes Ptp10D in this tumor-suppressive cell competition model  in the scrib mutant clones. The scrib−/− WASHY273D clones strongly resembled the overgrowth phenotype of scrib−/− Ptp10DRNAi clones  marked dividing cells than scrib−/− clones  overexpression also induced overgrowth of scrib−/− clones but the growth phenotype appeared milder compared to the scrib−/− WASHY273D clones . On the other hand, overexpression of a non-phosphorylatable form, WASHY273F27 or WASH knockdown by RNAi showed reduced wing sizes  promotes growth and inhibits apoptosis by activating proliferative and anti-apoptotic genes, such as expanded (ex) and Drosophila inhibitor of apoptosis-1 (Diap1). Hippo signaling controls growth by activating warts (wts), a kinase that directly phosphorylates and inhibits yki activity34. We examined the expression of ex-lacZ, a yki transcriptional reporter in the tumor-suppressive cell competition model35. Both the scrib−/−Ptp10DRNAi and scrib−/− WASHY273D clones showed elevated ex-lacZ levels compared to scrib−/−  driven WASHY273D overexpression , a transcriptional repressor downstream of EGFR pathway. Since Cic downregulation represents an upregulation of EGFR signaling41, we quantified the ratio of Cic signal in GFP positive and GFP negative cells. We detected similarly decreased Cic and upregulated EGFR signaling in scrib−/− WASHY273D and scrib−/− Ptp10DRNAi clones   showed defective apical localization of Ptp10D and punctuated Ptp10D, compared to cells in the control A-compartment  in a row and clustered photoreceptor recruiting cells posterior to MF48  and Vps29-RNAi ahead of morphogenic furrow but not in the scrib−/− clones  induces both yki and EGFR activation, and clone overgrowth. Future work is needed to directly investigate the presence of phosphorylated WASH in imaginal disc clones. Several studies have shown that the cytoskeletal tension imbalances lead to yki activation, and tissue growth33. These studies mainly focused on cortical F-actin filaments polymerization regulated by capping proteins, Diaphanous (Drosophila formin) and cofilin53. Our work shows that excessive endosomal F-actin polymerization may interfere with the cortical cytoskeleton and triggers yki activation, suppression of apoptosis and scrib−/− clone overgrowth. Proteomic analysis of retromer and retriever cargoes in human cell lines suggests that EGFR and PTPRJ (a human homologue of Ptp10D) are also recycled by different routes44. In our analysis of polarity-deficient epithelial cells, separation of EGFR from its negative regulator Ptp10D allowed increased EGFR signaling and subsequent tumorous growth. We speculate that in the subdomain of sorting endosome containing retriever cargos, EGFR may constantly stimulate Btk29A or other tyrosine kinases, which subsequently phosphorylate endogenous WASH. Phosphorylated WASH, boosts F-actin polymerization and presumably accelerates the basal level of retriever vesicles recycling directly to the plasma membrane. Conversely, in the sorting subdomain containing both retromer and retriever cargos, Ptp10D antagonizes EGFR or other RTKs. Thus, lower Btk29A activation leads to reduced-efficiency recycling by the retromer complex , UAS-WASHY273D27, UAS-Dicer2; engrailed-gal4::UAS-EGFP25752, Nubbin-gal486106, UAS-mCD8-GFP 5137, ex697 (ex-lacZ), UAS-bskDN 6409, UAS-RasV12 4847, UAS-EGFRDN 5364, UAS-Ptp10D-RNAi 39001, FRT82B2035 (Bloomington Drosophila Stock Center (BDSC)), UAS-WASH-RNAiv39769, UAS-Vps35-RNAiv45570, UAS-Vps29-RNAiv101375, UAS-Vps26- RNAiv18396, UAS-Arp2-RNAiv101999, UAS-Arp3-RNAiv108951 UAS-Btk29A-RNAiv106962, UAS-SNX27-RNAiv108542, UAS-SNX27-RNAiv28457, UAS-SNX17-RNAiv109452, UAS-SNX17-RNAiv43798, UAS-Vps26C-RNAiv39758, UAS-Vps26C-RNAiv104859, UAS-COMMD2-RNAiv109443, UAS-COMMD2-RNAiv27894, UAS-COMMD3-RNAiv16400, UAS-CCDC22-RNAiv109399, UAS-CCDC22-RNAiv36172, UAS-CCDC93-RNAiv35267, UAS-COMMD10-RNAiv15483, UAS-COMMD10-RNAiv15482 (Vienna Drosophila Research Center (VDRC)). All crosses were maintained at 25 °C.The following stocks are used: GFP labelled mitotic clones were induced in larval eye-antennal imaginal discs using the following strains:, y,w, eyFLP1;Act>y + >Gal4, UAS–GFP; FRT82B, Tub-Gal80 (82B tester-1), UAS-Dicer2; eyFLP5, Act>y + >Gal4, UAS-GFP; FRT82B, TubGal80 (82B tester-3), w Tub-Gal80, FRT19A; eyFLP5, Act>y + >Gal4, UAS- GFP (19 A tester) (kind gifts from Igaki’s lab). Additional strains used are as follows: 41 , Alexa FluorTM Phalloidin 647 , rabbit anti-ß-galactosidase , rabbit anti-dpERK  guinea pig anti-DIAP159 .Third instar larvae were dissected in 1xPBS and fixed in 4% paraformaldehyde (PFA) for 20 min and stained. For dpERK staining, third instar larvae were dissected in cold 1xPBS and immediate fixed in 8% PFA for 20 min and followed with 10 min ice-cold ethanol treatment in −20 °C before further step. Samples were incubated with primary antibodies at the following dilutions: chicken anti-GFP , rabbit anti-phospho-Histone H3 (Ser10) , rabbit anti-Dcp1 , mouse anti-WASH , P3H3) mouse anti-MMP1 , mouse anti-Ptp10D , rabbit anti-aPKCζ (C-20) , sc-216,), mouse anti-dEGFR , guinea pig anti-capicuag) for 10 min at 4 °C. The supernatant were further analyzed by SDS-PAGE gel. Blocking and antibody incubations were performed in TBST (TBS + 0.1% Tween 20). Samples were incubated with primary antibodies at the following dilutions: mouse anti-Ptp10D , mouse anti-dEGFR  and rabbit anti-tubulin . Three biological repeats were performed and calculated with Fiji.10 wing discs were dissected from third instar larvae and homogenized in 10 μl of RIPA buffer and Protease inhibitor cocktail tablets . The lysates were centrifuged at maximum speed . Clone sizes were measured as relative ratio (%) of GFP-positive area versus total area of eye disc based on DAPI in 2D projections of image Z-stacks using Fiji Software. The capicua signal was measured as ratio of intensities in GFP + /GFP- clones using Fiji Software. Statistical analysis was performed with the Graphpad Prism 7 software. Data in the scatter plot graphs were expressed as mean ± s.e.m. Two-tailed unpaired t-test, either Mann-Whitney U test or Welch’s t test was used to estimate statistical significance. Statistical significance was denoted as follows: n.s Further information on research design is available in the Supplementary InformationReporting Summary


Yohimbine (YOH) has antiproliferative effects against breast cancer and pancreatic cancer; however, its effects on vascular proliferative diseases such as atherosclerosis remain unknown. Accordingly, we investigated the inhibitory mechanisms of YOH in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF)-BB, a major mitogenic factor in vascular diseases. YOH (5–20 μM) suppressed PDGF-BB-stimulated a mouse VSMC line (MOVAS-1 cell) proliferation without inducing cytotoxicity. YOH also exhibited antimigratory effects and downregulated matrix metalloproteinase-2 and -9 expression in PDGF-BB-stimulated MOVAS-1 cells. It also promoted cell cycle arrest in the initial gap/first gap phase by upregulating p27Kip1 and p53 expression and reducing cyclin-dependent kinase 2 and proliferating cell nuclear antigen expression. We noted phospholipase C-γ1 (PLCγ1) but not ERK1/2, AKT, or p38 kinase phosphorylation attenuation in YOH-modulated PDGF-BB-propagated signaling pathways in the MOVAS-1 cells. Furthermore, YOH still inhibited PDGF-BB-induced cell proliferation and PLCγ1 phosphorylation in MOVAS-1 cells with α2B-adrenergic receptor knockdown. YOH (5 and 10 mg/kg) substantially suppressed neointimal hyperplasia in mice subjected to CCA ligation for 21 days. Overall, our results reveal that YOH attenuates PDGF-BB-stimulated VSMC proliferation and migration by downregulating a α2B-adrenergic receptor–independent PLCγ1 pathway and reduces neointimal formation in vivo. Therefore, YOH has potential for repurposing for treating atherosclerosis and other vascular proliferative diseases. Abnormal proliferation of vascular smooth muscle cells (VSMCs) is among the critical factors associated with vascular diseases such as atherosclerosis and restenosis ,2. In reVSMCs respond to PDGF-BB stimulation, which activates downstream signaling pathways and then drives cell cycle progression, cell proliferation, and cell migration . These bPausinystalia yohimbe and is a selective antagonist of α2-adrenergic receptors. It has been used in the treatment of erectile dysfunction for years. Yohimbine (YOH) can induce penile vascular smooth muscle contraction by selectively blocking presynaptic α2-adrenergic receptors [Yohimbine is an indole alkaloid derived from the bark of the African tree eceptors ,12. Moreeceptors ,14, antieceptors . In addieceptors  and can eceptors . Studieseceptors  and panceceptors . Moreoveeceptors ,21. Threeceptors ,23,24. Aeceptors . TherefoWe first determined the effects of YOH on PDGF-BB-induced VSMC proliferation by using the MTT assay, a typical assay to measure cellular metabolic activity as an indicator of cell viability . As illup < 0.001) and second gap (G2)/mitosis (M) phase ; however, the percentage of PDGF-BB-treated (10 ng/mL) MOVAS-1 cells in the initial gap (G0)/first gap (G1) phase decreased from 68.1 ± 0.8% to 52 ± 1.8%  VSMCs in the synthetic (S) phase /(Thr185/Tyr187) polyclonal antibody (pAb) was purchased from GeneTex , and anti-matrix metalloproteinase (MMP)-2 pAb was purchased from BioVision . Anti-MMP-9 pAb was purchased from Abcam . Anti-phospho-p38 MAPK (Thr180/tyr182) pAb and anti-p27Kip1 pAb were purchased from Affinity Biosciences . Anti-phospho-AKT mAb, anti-PCNA mAb, anti-CDK2 mAb, anti-phospho-PLCγ1 (Tyr783) pAb, and anti-phospho-(Ser) PKC pAb were purchased from Cell Signaling . Santa Cruz Biotechnology -produced anti-p53 mAb and anti-α2B-adrenergic receptor mAb were utilized. Enhanced chemiluminescence Western blotting detection reagent and polyvinylidene difluoride (PVDF) membranes were from GE Healthcare Life Sciences . Yohimbine hydrochloride was dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C until use.Yohimbine hydrochloride was purchased from Cayman Chem . Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine/penicillin/streptomycin, trypsin (0.25%), and anti-α-tubulin monoclonal antibody (mAb) were purchased from Thermo Fisher Scientific . Moreover, recombinant Human PDGF-BB (carrier-free) was purchased from BioLegend . RNase A was purchased from New England Biolabs  and 3--2,5-diphenyltetrazolium bromide (MTT) was purchased from Merck . Horseradish-peroxidase-conjugated donkey antirabbit IgG and sheep antimouse IgG were purchased from Amersham . Anti-phospho-ERK1/2  lentiviral particles purchased from Santa Cruz Biotechnology were added to the culture. The MOVAS-1 cells were seeded in a six-well plate at a density of 1.5 × 105 cells/well and then transfected with shRNA lentiviral particles mixed with Polybrene in Opti-MEM  for 24 h. To select stable clones, the medium was replaced with fresh puromycin-containing medium (10 μg/mL) every 3 days.A murine vascular smooth muscle cell line (MOVAS-1) was purchased from ATCC . Cells were cultured in DMEM supplemented with 10% FBS, penicillin G (100 units/mL), streptomycin (100 mg/mL), and glutamine (2 mM) at 37 °C in a humidified atmosphere of 5% CO4 cells/well) and cultured in DMEM with 10% FBS for 24 h. The medium was then replaced with serum-free medium. The cells were pretreated with YOH  for 45 min and then stimulated with or without PDGF-BB (10 ng/mL) for 48 h. Cell viability was measured using a colorimetric assay based on the ability of mitochondria in viable cells to reduce MTT into purple formazan, as described previously [MOVAS-1 cells were seeded in 24-well plates  for 45 min and then stimulated with PDGF-BB (10 ng/mL) for 24 h. Images of the migrated area were captured by a microscope  and analyzed using ImageJ.6 cells/dish) were pretreated with YOH (10 or 20 μM) for 45 min and then stimulated with or without PDGF-BB (10 ng/mL) for 18 h. After 18 h, the cells were collected using trypsin, washed with cold PBS, and then fixed in ice-cold 70% ethanol overnight. The cell samples were then washed with PBS and resuspended in a PI solution  for 30 min and subjected to a flow cytometry test .In the cell cycle analysis, starved MOVAS-1 cells  and then subjected to a gel electrophoresis system. The protein samples were loaded and separated on sodium dodecyl sulfate–polyacrylamide gel. After 1.5 h, the separated proteins were transferred to PVDF blotting membranes by semi-dry electrophoretic transfer cell . The transferred membranes were incubated with BlockPRO blocking buffer  for 1 h and then with different primary antibodies for 2 h, after which they were incubated with a secondary antibody for 1 h. An ECL system was used to detect immune-reactive bands in this study. Densitometry was performed to quantify protein bands by using Visionworks for Windows .A mouse model of common carotid artery (CCA)-ligation–induced neointimal hyperplasia was applied as previously described, with some modifications . This prn). Data were first assessed using one-way analysis of variance (ANOVA). If the one-way ANOVA revealed significant differences among the group means, then the Newman–Keuls method was applied subsequently for comparison. A p value of <0.05 was considered to indicate a statistically significant difference.The results are presented as the mean ± standard error (S.E.M.) and the number of independent experiments (


We engineered a non-replicating pseudotyped influenza virus (S-FLU) to deliver the well-known cancer testis antigen, NY-ESO-1 (NY-ESO-1 S-FLU). Intranasal or intramuscular immunization of NY-ESO-1 S-FLU virus in mice elicited a strong NY-ESO-1-specific CD8+ T cell response in lungs and spleen that resulted in the regression of NY-ESO-1-expressing lung tumour and subcutaneous tumour, respectively. Combined administration with anti-PD-1 antibody, NY-ESO-1 S-FLU virus augmented the tumour protection by reducing the tumour metastasis. We propose that the antigen delivery through S-FLU is highly efficient in inducing antigen-specific CD8+ T cell response and protection against tumour development in combination with PD-1 blockade.Virus-based tumour vaccines offer many advantages compared to other antigen-delivering systems. They generate concerted innate and adaptive immune response, and robust CD8 The fight against cancer remains unfinished as it continues to be a major threat to human life. Tumour antigen-specific strategies such as the dendritic cell (DC) vaccine  have beeIntrinsically immunogenic pathogens with potential pattern recognition receptor (PRR) agonistic functions can induce potent anti-tumour responses , and, wiInfluenza A virus (IAV) is an interesting candidate for antigen delivery since IAV infection elicits a strong antigen-specific CTL response. We developed previously a pseudotyped replication-deficient influenza A/Puerto Rico/8/34 (PR8) virus, which replicates only in the cell line expressing HA that provides the HA protein on pseudotyped virus particles for their binding to cells ; importa+ lung epithelial cells compared to immune cells (identified as CD45+)  .The ability of intranasal infection with NY-ESO-1 S-FLU virus to induce a T cell primary activation/T cell priming was then investigated in draining lymph nodes (dLN). Using syngeneic 4T1 metastatic breast carcinoma (MBC) tumour model, we tested NY-ESO-1 S-FLU virus anti-tumour effect in the following settings: (1) prophylactic, (2) therapeutic, and (3) spontaneous tumour metastasis models. In prophylactic tumour model, immunization with NY-ESO-1 S-FLU virus significantly reduced the number of nodules in lungs. Interestingly, a single infection with NY-ESO-1 S-FLU was sufficient to give significant protection against tumour challenge . In the + T cell response in lungs, intranasal infection did not elicit a high CTL response in spleen  and NY-ESO-1 (p<0.05)-specific CD8+ T cell response compared with intramuscular injection. In the blood and spleen, intramuscular infection induced stronger NP (p<0.01) and NY-ESO-1 (p<0.01)-specific CTL responses than intranasal infection (+ T cells (triple producers) was observed in HHD mice spleen following intramuscular infection  has been implicated in the retention of T cells  were significantly upregulated on NY-ESO-1-specific CD8+ T cells in the lungs of intranasally infected mice (+ T cells (TRM) (p<0.0001) were also significantly higher in intranasally infected mice  implicated in retention or trafficking of effector CD8ted mice  Furthermted mice .+ T cell responses in lungs or spleen were analysed on day 7. NY-ESO-1- and NP-specific CTL responses were reduced in lungs following the infection with NY-ESO-1 S-FLU virus with matched HA/NA  virus to elicit the antibody (against H3N2) production, and on day 24 post infection, mice were reinfected with NY-ESO-1 S-FLU virus with matched HA/NA (NY-ESO-1 S-FLU [X31]) or mismatched HA/NA (NY-ESO-1 S-FLU [H1 PR8]). NY-ESO-1-specific CD8ed HA/NA . Similared HA/NA .+ T cells in TIL (p<0.01)  and (p<0.01 on days 26 and 28), which was concomitant with significantly higher infiltration of NY-ESO-1-specific CD8(p<0.01) , whereas(p<0.01) . We alsoted mice . In clonted mice . However3) compared to the mice treated with Hu Ad5-NY-ESO-1 (972.0 ± 90.6 mm3) or Fowl Pox-NY-ESO-1 (1137.4 ± 223.6 mm3) viruses . HHD mice showed a robust NY-ESO-1-specific CTL response following infection with NY-ESO-1 S-FLU virus , and for+ T cells into the tumour in NY-ESO-1 S-FLU virus-infected mice + T cell response  is one of the strongest inducers of CD8response , and theresponse . Intrana in humans (+ T cells from the tumour site (skin) to distant site (lungs), resulting in decreased immunity within the tumour  and PD-1 antibody-expressing S-FLU  could clear the tumour completely .157-165-specific TCR (Human leukocyte antigen (HLA)-A2.1 transgenic mice   was optimized by removal of unwanted ATG sequences. GFP S-FLU was made similarly with GFP replacing the NY-ESO-1 sequence. Recombinant NY-ESO-1 S-FLU on the A/PR/8/1934 background were produced by transfection of HEK 293T cells as described (g for 5 min) of the culture supernatant to remove debris and kept as a seed virus. NY-ESO-1 S-FLU (X31) was generated by infecting MDCK-SIAT1 stably transfected with X31 H3 (MDCK-X31) with the NY-ESO-1 S-FLU (PR8) seed virus (approximately multiplicity of infection [MOI] 0.01) and the supernatant was harvested as described above after 48 hr. NY-ESO-1 S-FLU was titrated as TCID50 as previously described . Media were supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine and penicillin/streptomycin. NY-ESO-1 S-FLU was generated as previously described  with minescribed  and clonescribed . In brieescribed .The full-length NY-ESO-1 gene (GenBank no: NM001327) was cloned into the replication-deficient human adenovirus serotype 5 (AdHu5) vector backbone under the control of the human CMV immediate early long promoter to generate the HuAd5-NY-ESO-1 construct. The replication-deficient adenoviral vectors were scaled up by the Viral Vector Core Facility at the Jenner Institute  in 293A cells with purification by caesium chloride centrifugation and stocks were stored at –80°C in PBS. Virus titre was determined in a cytopathic effect assay . Purity 6 TCID50 of GFP S-FLU, 1 × 106 or 3 × 106 TCID50 of NY-ESO-1 S-FLU (PR8) virus or NY-ESO-1 S-FLU (X31) virus and 3.2 × 104 TCID50 of X31 virus. For intramuscular route, 6 × 106 TCID50 (100 μl volume) of NY-ESO-1 S-FLU (PR8) virus was used under inhalation isoflurane anaesthesia. In some experiments, intramuscular infection with 1 × 109 PFU of Hu Ad5 NY-ESO-1 virus or 5 × 107 PFU of Fowl Pox NY-ESO-1 virus was also performed.Influenza virus infections via intranasal route (50 μl volume) were performed with 1 × 10+ T cells from 1G4 mice spleen were enriched using MACS beads  and labelled with 5 μM CellTrace Violet (CTV) (Thermo Fisher Scientific) following the manufacturer’s instructions. Approximately 2 × 106 CD8+ T cells in 200 μl volume were adoptively transferred to HHD mice.Single-cell preparation from lungs was prepared as described before . Briefly6) were isolated from either naïve or infected or tumour-bearing HHD or BALB/c mice and were cultured in the presence of HLA-A2-restricted IAV M1 peptide 158-66(GILGFVFTL) (Cambridge peptide) or NY-ESO-1 peptide 157-65(SLLMWITQC) (Sigma peptide) or H-2Kd binding IAV NP peptide 147-55(TYQRTRALV) (Cambridge peptide) or H-2Dd-restricted NY-ESO-1 peptide 81-88(RGPESRLL) (Cambridge peptide) in complete RPMI 1640 medium supplemented with 10% FBS, 2.1 mmol/l ultra-glutamine in the presence of brefeldin A (5 μg/ml) and monensin . After 5 hr of incubation, cells were stained for extracellular markers  and viability dye Near IR dead cell staining kit, Invitrogen. Cells were then stained for intracellular IFNγ, TNFα, and IL2 using an Intracellular Fixation and Permeabilization Buffer Set (eBioscience) following the manufacturer’s instructions. In some experiments, lung cells or splenocytes were prepared as described above and stained with appropriate surface antigens followed by PE-labelled H-2Dd-restricted NY-ESO-1 peptide 81-88(RGPESRLL) and brilliant violet 421-labelled HLA-A2-restricted IAV M1 peptide 158-66(GILGFVFTL) or H-2Kd-binding IAV NP peptide 147-55(TYQRTRALV) (kindly provided by NIH tetramer facility at Emory University). Samples were acquired on a FACScanto II or LSR Fortessa-X50 flow cytometer (BD Biosciences), and data were analysed with FlowJo version 10.4.1. Compensation beads (eBioscience) were used to generate the compensation matrix, and FMOs were used as control.Splenocytes or lung cells  mice were euthanized and lungs were fixed in Bouin’s solution to count the tumour nodules. For therapeutic tumour model, mice were first injected with 2 × 105 4T1-NY-ESO-1 cells on day 0 followed by intranasal infection with GFP S-FLU or NY-ESO-1 S-FLU virus on days 2 and 10 in the doses mentioned above. Mice were closely monitored and euthanized on day 16 post tumour cell injection. For the spontaneous metastasis model, 4T1 cells were subcutaneously injected into the flank of BALB/c mice and the mice were intranasally or intramuscularly infected with GFP S-FLU or NY-ESO-1 S-FLU virus on days 4 and 18 post tumour cell injection. In some experiments, immunized mice also received intraperitoneal injection of anti-PD-1 antibody  or isotype antibody. Tumour volumes were measured every other day until days 28–30 at humane end point (when tumour size reaches >1.2 cm3). Then mice were euthanized and metastasis colony formation assay (clonogenic assay) was performed to quantify 4T1-derived cells in the lungs of transplanted mice. Entire lungs were minced and incubated in HBSS media with 10% FBS, supplemented with collagenase  and deoxyribonuclease  for 45 min. Thereafter, lung fragments were homogenized through a 100 μm filter and rinsed with 5 ml of PBS. After centrifugation, cell pellets were subjected to RBC lysis , and after two times washing in PBS, cells were resuspended in 10 ml of selection media (RPMI 1640 media with 10% FBS and 60 μM 6-thioguanine (Sigma-Aldrich)) before being diluted 1:10 and 1:100 in selection medium. Cells were plated in culture dishes, and the colonies were stained after 14 days with 1% crystal violet/70% methanol solution and counted.For tumour model, 4T1 MBC and 1F4 (tumour cells generated from methylcholanthrene-induced tumours from HHD mice) – syngeneic mouse models have been used as described before  with som5 NY-ESO-1-expressing tumour cells (1F4) by subcutaneous injection and tumour size was monitored until humane end point.To analyse whether memory T cell response elicited by NY-ESO-1 S-FLU infection protects mice against tumour challenge, HHD mice were infected on day 0 with GFP-S FLU virus or NY-ESO-1 S-FLU virus via intramuscular route and/or NY-ESO-1 S-FLU virus via intranasal route. Mice received booster infection with appropriate virus on day 14 post primary infection. On day 30 post booster infection, mice were challenged with 5 × 10t-test and one-way ANOVA with Tukey’s multiple comparisons were used to calculate statistical significance .Unpaired, two-tailed Student’s  The authors found out that virus-based tumour vaccines can induce a robust CTL response capable of limiting tumour progression, which is interesting to many researchers who are looking for ways to enhance CTL response to tumour immunity in combination with checkpoint inhibitors. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Päivi Ojala as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Recombinant single-cycle influenza virus with exchangeable pseudo types, allows repeated immunisation to augment antitumour immunity with immune checkpoint inhibitors" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) In Figure 6, what are the mechanisms of superior efficacy of S-Flu-based vaccine compared with Ad5 or Pox-based vaccine? Are quantity and/or quality of NY-ESO1-specific CTL different depending on the kinds of virus vectors? Alternatively, other types of immune cells may be also stimulated by each virus vector.2) The authors should perform additional experiments in which they examine tumor volume following challenge of mice that were influenza infected 30 or more days previously. This experiment would allow the authors to determine the extent to which the reduction in tumor volume seen in their current experiments following challenge infection is due to an ongoing effector response. This is an important proof of concept that a recombinant influenza infection approach generates a memory T cell response capable of eliciting protective immunity against tumor challenge.3) The author should also assess whether PD1 blockade is able to reduce tumor development when administered to mice that were influenza infected 30 or more days previously and then challenged. This would determine whether PD1 blockade enhances the functionality of memory T cells following recombinant influenza infection. Currently, the authors only perform PD1 blockade beginning 4 days after influenza infection.eLife.Both reviewers recognize that this manuscript is potentially interesting to tumor-immunity researchers. But, reviewers request the experiments  to make the impact strong enough for publication in Reviewer #1 (Recommendations for the authors):1. It is extremely important to enhance the efficacy of ICI with vaccination, while some clinical trials using peptide-based CT antigen vaccine failed to show its synergistic effect. Thus, Flu-based vaccines seem to be a promising strategy to enhance the effect of ICI. However, it is unclear whether the synergistic effect of S-Flu-NYESO1 is not usually observed in pre-clinical studies using other types of vaccine. The authors are recommended to discuss these issues.2. Is the expression level of NY-ESO1 in 4T1-NY-ESO1 cells comparable to those expressed in primary tumor cells? This may be a difficult question to answer, but the authors are recommended to discuss whether NY-ESO1 is expressed at physiological levels. Essential revisions:1) In Figure 6, what are the mechanisms of superior efficacy of S-Flu-based vaccine compared with Ad5 or Pox-based vaccine? Are quantity and/or quality of NY-ESO1-specific CTL different depending on the kinds of virus vectors? Alternatively, other types of immune cells may be also stimulated by each virus vector.It is an interesting question and thank you for your observation. A significantly higher CTL response with S-FLU virus compared to POX virus explains the enhanced anti-tumour effect of S-FLU over Pox virus based vaccine. However, we don’t know the clear reason for the superior anti-tumour efficacy of S-FLU -NY-ESO1 over Ad5 based vaccine. The frequencies of NY-ESO-1 specific CTL responses in lungs and spleen were similar following S-FLU-NY-ESO1 or Hu-Ad5 NY-ESO1 administration. We haven’t investigated the quality or polyfunctionality of CTLs and differential cellular infiltration to the tumour site post immunization with S-FLU or Ad5virus. S-FLU and Ad5 virus elicit different kinds of activation of innate signalling  and we surmise that these different innate immune signalling may influence subsequent immune response including cellular infiltration.2) The authors should perform additional experiments in which they examine tumor volume following challenge of mice that were influenza infected 30 or more days previously. This experiment would allow the authors to determine the extent to which the reduction in tumor volume seen in their current experiments following challenge infection is due to an ongoing effector response. This is an important proof of concept that a recombinant influenza infection approach generates a memory T cell response capable of eliciting protective immunity against tumor challenge.Thank you for your suggestion. We investigated whether the memory T cell response can elicit protective immunity against tumour challenge in HHD mice. We have included the result as supplementary Figure 10 and updated the text in manuscript.3) The author should also assess whether PD1 blockade is able to reduce tumor development when administered to mice that were influenza infected 30 or more days previously and then challenged. This would determine whether PD1 blockade enhances the functionality of memory T cells following recombinant influenza infection. Currently, the authors only perform PD1 blockade beginning 4 days after influenza infection.Both reviewers recognize that this manuscript is potentially interesting to tumor-immunity researchers. But, reviewers request the experiments  to make the impact strong enough for publication in eLife.Thank you for your observation and excellent suggestion. Unfortunately, our lab has been closed because of our PI’s ( Vincenzo Cerundolo ) untimely death. Because of that and on-going COVID-19 pandemic related limitations, we managed to do the experiment for memory T cell response only without PD-1 co-administration.Reviewer #1 (Recommendations for the authors):1. It is extremely important to enhance the efficacy of ICI with vaccination, while some clinical trials using peptide-based CT antigen vaccine failed to show its synergistic effect. Thus, Flu-based vaccines seem to be a promising strategy to enhance the effect of ICI. However, it is unclear whether the synergistic effect of S-Flu-NYESO1 is not usually observed in pre-clinical studies using other types of vaccine. The authors are recommended to discuss these issues.Enhanced anti-tumour effect of virus-based vaccine co-administered with ICI has been recently reported for adenovirus based vaccine : e003218) and S-FLU-NY-ESO-1 virus has not been investigated before for anti-tumour therapy and in this study, we report the synergistic effect of S-FLU-NY-ESO-1 with immune check point inhibitor for the first time. We included these issues in discussion.2. Is the expression level of NY-ESO1 in 4T1-NY-ESO1 cells comparable to those expressed in primary tumor cells? This may be a difficult question to answer, but the authors are recommended to discuss whether NY-ESO1 is expressed at physiological levels.157-165 Ags per cell in which density most self Ags and TAAs are presented :7308-16). We used 4T1 tumour cells with lower level of NY-ESO-1 expression for our tumour study since the higher expression of NY-ESO-1 in tumour cells resulted in spontaneous tumour rejection. We included this information in discussion.The level of NY-ESO-1 expression may vary in different types and different stages of tumour. The physiological level of NY-ESO-1 expression in melanoma is an average of ∼10-50 NY-ESO-1


Autism spectrum disorder (ASD) is a common neurodevelopmental disorder, with an increasing prevalence worldwide. Copy number variation (CNV), as one of genetic factors, is involved in ASD etiology. However, there exist substantial differences in terms of location and frequency of some CNVs in the general Asian population. Whole-genome studies of CNVs in Northeast Han Chinese samples are still lacking, necessitating our ongoing work to investigate the characteristics of CNVs in a Northeast Han Chinese population with clinically diagnosed ASD. We performed a genome-wide CNVs screening in Northeast Han Chinese individuals with ASD using array-based comparative genomic hybridization. DRD4, HRAS, OPHN1, SHANK3, SLC6A3, and TSC2] and two deletions [CHRNA7 and PTEN]) and 12 microRNAs-coding genes . We found that 22 kinds of CNVs (6 deletions and 16 duplications) were potentially pathogenic. These CNVs were distributed in chromosome 1p36.33, 1p36.31, 1q42.13, 2p23.1-p22.3, 5p15.33, 5p15.33-p15.2, 7p22.3, 7p22.3-p22.2, 7q22.1-q22.2, 10q23.2-q23.31, 10q26.2-q26.3, 11p15.5, 11q25, 12p12.1-p11.23, 14q11.2, 15q13.3, 16p13.3, 16q21, 22q13.31-q13.33, and Xq12-q13.1. Additionally, we found 20 potential pathogenic genes of ASD in our population, including eight protein coding genes (six duplications [We identified CNVs and genes implicated in ASD risks, conferring perception to further reveal ASD etiology.The online version contains supplementary material available at 10.1186/s12888-023-04565-7. CHD8 CHD8 , or AutismKB [http://www.autismkb.com]); (4) they had been found in the Database of genomic variation and phenotype in Humans using Ensembl Resources ; and (5) they contained previously reported ASD-relative genes.CNVs were considered of strong putative interest when they reached the following criteria: (1) they were classified as likely pathogenic or pathogenic; (2) they were of large size (> 100 kb); (3) they had been found in the knowledgebases for the genetic evidence of ASD  genes enriched in ASD-related pathways; and (2) same genes shared with 363 genes in SFARI classified as high-confidence or strong-candidate, or with 228 genes in AutismKB classified as high-confidence.MicroRNAs (miRNAs) are involved in the pathogenesis of ASD , 36. BecP-value < 0.05 was considered statistically significant. miRWalk 2.0 database, which contained 12 miRNA-target-prediction database, was used to predict target genes of CNVs-encoded miRNAs  and two deletions [MIR107 and MIR558]). BDNF, a brain-derived neurotrophic factor and a member of the neurotrophic factor family, is a target gene of miR-202 [BNDF. Skogstrand et al. found that lower BDNF levels in serum correlate with ASD risks [DIP2A and ASD risks has been validated by our team [Dip2a knockout mice exhibit autism-like behaviors, including excessive repetitive behavior and social novelty defects [MECP2 transgenic monkeys corroborate association between miR-339-5p and MECP2 [MECP2. For these reasons, miRNAs encoded within CNVs may be implicated in ASD etiology.MiRNAs encoded within CNVs are important functional variants, providing a new dimension to recognize the association between genotype and phenotype . MiRNAs SD risks . miR-339SD risks , 71. In our team ; moreove defects . Notablynd MECP2 . In addiFor enrichment analysis, we found that genes were enriched in synapse, synapse-related signal regulation, neurotransmitter activity, neurotransmitter transport, and neurotransmitter binding. Mutations in synapse-related or neurotransmitter-related genes are associated with ASD risks in multiple unbiased, targeted sequencing, and neuropathological studies, evidencing that dysregulation in synaptogenesis and neurotransmission is implicated in the pathogenesis of ASD –78. We cFunctional network analysis of the 219 CNVs-encoded-miRNAs-targeted genes elicited that a novel regulating mechanism of these CNVs-encoded miRNAs consisted of synapse-related functions , axon guidance, ion channel (ion-gated channel and cation channel complex), and Wnt signaling pathway. Synaptic function and Wnt signaling pathway are affected by mutations in diverse ASD-related genes, and altered Wnt pathway signaling may confer an involvement in ASD pathogenesis . InteresBoth GO and Pathway enrichment analyses showed that CNVs-relating genes and CNVs-encoded-miRNAs-targeted genes mapped synapse-related functions. Additionally, CNVs-relating genes also enriched in mTOR signaling pathway and insulin signaling pathway. In contrast, CNVs-encoded-miRNAs-targeted genes enriched in axon guidance, ion channel, and Wnt signaling pathway. These results documented the high complexity and heterogeneity of ASD, suggesting that different genomic alteration on same chromosomal location may confer distinct but complementary effects on the brain of people with ASD.Our study had some limitations: (1) the sample size in our study may confer limited statistical power to discover significant findings; (2) genetic and environmental factors contribute to ASD risk; however, environmental factors were not available for us; and (3) de novo or inherited of the CNVs were not be classified because of the lack of data from parents.Despite these limitations above, our study also had some strength. Firstly, we found eight de novo CNVs  and 12 validated CNVs , further documenting that ASD is of high genetic heterogeneity after comparing our results and previous findings , eight protein-coding genes, and 12 miRNAs-coding genes are implicated in ASD risks, conferring perception to further reveal ASD etiology.Additional file 1. Supplementary Table 1. Summary of potential Pathogenic CNVs of ASD. Supplementary Table 2. Functional annotation  of the 511 genes from 22 potential pathogenic CNVs (top 20). Supplementary Table 3. Functional annotation (cellular components) of the 511 genes from 22 potential pathogenic CNVs (top 20). Supplementary Table 4. Functional annotation (molecular function) of the 511 genes from 22 potential pathogenic CNVs (top 20). Supplementary Table 5. Pathway enrichment of the 511 genes from 22 potential pathogenic CNVs (top 20). Supplementary Table 6. CNVs-encoded-miRNAs-target genes of ASD (duplication). Supplementary Table 7. CNVs-encoded-miRNAs-target genes of ASD (deletion). Supplementary Table 8. Functional annotation  of the 219 target genes of potential pathogenic miRNAs coded within CNVs (top 20). Supplementary Table 9. Functional annotation (cellular components) of the 219 target genes of potential pathogenic miRNAs coded within CNVs (top 20). Supplementary Table 10. Functional annotation (molecular function) of the 219 target genes of potential pathogenic miRNAs coded within CNVs (top 20). Supplementary Table 11. Pathway enrichment of the 219 target genes of potential pathogenic miRNAs coded within CNVs (top 20). Supplementary Table 12. Comparison of CNVs involved in ASD. Supplementary Fig. 1. Venn diagram based on ASD_SFARI, ASD_AutismKB, and CNVs-encoded-miRNAs-targeted genes.


After false-discovery rate correction, ASB was genetically correlated with COVID-19  and 19 other traits. ASB and COVID-19 were both positively genetically correlated with having a noisy workplace, doing heavy manual labor, chronic obstructive pulmonary disease, and genitourinary diseases. ASB and COVID-19 were both inversely genetically correlated with average income, education years, healthspan, verbal reasoning, lifespan, cheese intake, and being breastfed as a baby. But keep in mind that rgs are not necessarily causal. And, if causal, their prevailing directions of effect (which causes which) are indiscernible from rgs alone. Moreover, the SNP-heritability (Little is known about the genetics of norm violation and aggression in relation to coronavirus disease 2019 (COVID-19). To investigate this, we used summary statistics from genome-wide association studies and linkage disequilibrium score regression to calculate a matrix of genetic correlations ( Antisocial behavior (ASB)—including rule-breaking and violence—is harmful to society. ASB creates a long wake of monetary and emotional disturbances for countries, communities, and individuals , 2. Espergs) [rgs between ASB and most psychiatric, reproductive, cognitive, and addictive traits [rg = −0.55) between ASB and self-reported health has been reported [rgs between ASB and 669 health, physiological, and well-being measures after accounting for multiple testing [Complicating causal inference concerning ASB and pandemic-relevant outcomes is that about half of the variance in ASB and, to varying degrees, associated traits, is heritable –9. This rgs) . These se traits , 12. In e traits , 14. A sreported . In cont testing . Thus, mhttp://www.github.com/bulik/ldsc) [rgs. Of note is that correlation, even when genetic, is not necessarily causation. While our study can point to shared genetic architecture between traits, the reader should be cautious about assuming that the rgs are causal. Table We characterized the shared polygenic nature of ASB, COVID-19, and selected health and behavioral traits using summary statistics from GWA studies and linkage disequilibrium score regression  . We calcP < 0.05), ASB was positively genetically correlated with COVID-19 (release 4): rg = 0.51; P = 1.54E-02. The rg was also positive between ASB and COVID-19 (release 6) with marginal significance prior to FDR-correction but not after: rg = 0.35; P = 3.83E-02 (FDR-corrected P = 5.21E-02). The remaining (FDR-significant) rgs between ASB and health and behavioral traits that were positively genetically correlated are as follows:rg = 0.70)Townsend Deprivation Index (rg = 0.63)Noisy workplace (rg = 0.58)Heavy manual labor (rg = 0.51)COPD (rg = 0.50)Risk tolerance (rg = 0.46)Gastrointestinal diseases (rg = 0.42)Seen a doctor for nerves, anxiety, tension, or depression (rg = 0.42)Seen a sudden violent death (rg = 0.38)Genitourinary diseases (rg = 0.36)Being a violent-crime victim (rg = 0.29)Neuroticism (rg = 0.15)Playing computer games -correction (rg = -0.54)Average income (rg = -0.48)Education years (rg = -0.47)Healthspan (rg = -0.44)Verbal reasoning (rg = -0.33)Lifespan (rg = -0.28)Cheese intake (rg = -0.24)Breastfed as baby :rgs for ASB and the health and behavioral traits are displayed in a forest plot in Fig. P-values before and after FDR-correction  -- COVID-19 (release 6)COPD (rg = 0.33) -- COVID-19 (release 4)COPD (rg = 0.38) -- COVID-19 (release 6)Heavy manual labor (rg = 0.20) -- COVID-19 (release 4)Heavy manual labor (rg = 0.32) -- COVID-19 (release 6)Genitourinary diseases (rg = 0.28) -- COVID-19 (release 6)Noisy workplace (rg = 0.26) -- COVID-19 (release 4)Noisy workplace (Due to the positive rg = -0.39) -- COVID-19 (release 6)Cheese intake (rg = -0.36) -- COVID-19 (release 4)Cheese intake (rg = -0.46) -- COVID-19 (release 6)Education years (rg = -0.32) -- COVID-19 (release 4)Education years (rg = -0.49) -- COVID-19 (release 6)Verbal reasoning (rg = -0.28) -- COVID-19 (release 4)Verbal reasoning (rg = -0.41) -- COVID-19 (release 6)Healthspan (rg = -0.25) -- COVID-19 (release 4)Healthspan (rg = -0.24) -- COVID-19 (release 6)Breastfed as baby (rg = -0.30) -- COVID-19 (release 6)Lifespan (rg = -0.21) -- COVID-19 (release 6)Average income  , Carvalhrgs between ASB and the psychiatric and violence-related traits we measured. But none of these traits were genetically correlated with COVID-19. That they were not comports with a meta-analytic review of mood disorders and risk for COVID-19 in 91 million individuals [We observed positive ividuals . Namely,ividuals . Thus, trg for ASB and COVID-19 dropped from 0.51 (release 4) to 0.35 (release 6). Earlier GWA study releases by the COVID-19 Host Genetics Initiative capture data from earlier timepoints in the pandemic—release 4 being earlier  than release 6 . This may be important since release 4 occurred before vaccines against SARS-CoV-2 were available, and by June 15, 2021, 47% of those eligible for vaccination had completed an initial protocol for full vaccination in the U.S [We note that the strength of the  the U.S . Also, b October , 2020 thrgs between ASB and COVID-19 were considerable, in absolute terms the genetic variance that is overlapping between the two traits is, likewise, small. Second is that rgs, while robust against most environmental confounders, can still suffer from genetic sources of confounding . To illustrate, it seems unlikely that not being breastfed as a baby and eating less cheese cause ASB. One should, for somewhat obvious reasons, be similarly cautioned against the conclusion that being breastfed as a baby and eating more cheese protect against COVID-19, despite the significant rgs. Indeed, we chose these dietary traits to communicate the point that the shared genetic architectures that these have with education years, verbal reasoning, and average income seem the more plausibly causal phenomena. Third, supposing that some of the rgs represent causal linkages in some way, we nonetheless cannot determine the direction of causality with rgs alone. For much of the discussion above, we tacitly presumed plausible directions of effect . But with all the traits in our matrix, the prevailing direction of effect could be the opposite and/or some level of bi-directional causation may exist [rgs obtained from LDSC are not affected by sample overlap  [Our study has limitations, which must also be considered. First is that the ay exist , 21–23. ay exist  models tculated) . This isSupplement