key: cord-0277830-nh2muq2d authors: Diray-Arce, J.; Angelidou, A.; Jensen, K. J.; Conti, M. G.; Kelly, R.; Pettengill, M. A.; Liu, M.; van Haren, S. D.; McCulloch, S.; Michelotti, G.; the EPIC Consortium,; Kollmann, T.; Kampmann, B.; Steen, H.; Ozonoff, A.; Lasky-Su, J.; Benn, C. S.; Levy, O. title: Bacille Calmette-Guerin vaccine reprograms human neonatal lipid metabolism in vitro and in vivo date: 2021-12-05 journal: nan DOI: 10.1101/2021.12.03.21267251 sha: 19bb56fda1d6ade4a8f4accc27719bdf0bec52a5 doc_id: 277830 cord_uid: nh2muq2d Vaccines have generally been developed with limited insight into their molecular impact. While systems vaccinology, including metabolomics, enables new characterization of vaccine mechanisms of action, these tools have yet to be applied to infants at high risk of infection and receive the most vaccines. Bacille Calmette-Guerin (BCG) protects infants against disseminated tuberculosis (TB) and TB-unrelated infections via incompletely understood mechanisms. We employed mass spectrometry-based metabolomics of blood plasma to profile BCG-induced infant responses in Guinea Bissau in vivo and the U.S. in vitro. BCG selectively altered plasma lipid pathways, including lysophospholipids. BCG-induced lysophosphatidylcholines (LPCs) correlated with both TLR agonist- and purified protein derivative (PPD, mycobacterial antigen)-induced blood cytokine production in vitro, raising the possibility that LPCs contribute to BCG immunogenicity. Analysis of an independent newborn cohort from The Gambia demonstrated shared vaccine-induced metabolites such as phospholipids and sphingolipids. BCG-induced changes to the plasma lipidome and LPCs may contribute to its immunogenicity and inform the discovery and development of early life vaccines. To elucidate the impact of early BCG vaccination, plasma samples from in vivo and in 147 vitro cohorts were subjected to comprehensive metabolomics analysis. Our primary in vivo 148 cohort consisted of low birthweight newborns from Guinea-Bissau who were enrolled in a 149 randomized clinical trial to receive BCG at birth (classified as early BCG) or at six weeks after 150 birth (delayed BCG) (Biering-Sorensen et al., 2017). In an immunological study nested within 151 the trial, capillary blood samples were collected four weeks after randomization (after BCG was 152 given in the early BCG group and before BCG was given in the delayed BCG group) to 153 investigate the effect of BCG on PPD antigen-and TLR-agonist-induced whole blood cytokine 154 responses in vitro ( Figure 1A ) (Jensen et al., 2015) . Catch-up vaccination followed at six weeks 155 of life with Pentavalent vaccine (Penta) and oral polio vaccine (OPV) for the early BCG group 156 and BCG, Penta, and OPV for the delayed BCG group (Figure 2A) . contrast, early vs. late BCG administration was associated with lower plasma amino acid 189 concentrations ( Figure 2D ). 190 191 BCG induced prominent shifts in plasma lipid pathways 192 Compared to the delayed BCG group, early BCG administration was associated with 193 distinct concentrations of a range of lipid pathways and metabolites. Of note, greater than half of 194 the metabolites whose levels changed significantly (p< 0.05) between the groups belonged to the 195 lipid superclass ( Figure 3A ), and 39% of DAMs were of the lysophospholipid (LPL) subclass 196 ( Figure 3A ). Early BCG administration was consistently associated with robust production of 197 selected sphingolipid, monoacylglycerol, steroid, and LPL metabolites (Table S2 ). In contrast, 198 palmitoylglycerols and pregnanediol disulfate (progestin steroids) were decreased in the early vs. 199 delayed BCG group ( Figure 3B , Table S2 ). 200 201 We used pathway-based network reconstruction (implementation: Metscape plugin of 202 Cytoscape) to construct a compound network providing a comprehensive overview of metabolic 203 signatures to compare early vs. delayed BCG newborn groups ( Figure 3C ). As BCG 204 administration at birth was associated with high production of lipid metabolites, our network 205 demonstrated that glycerolipid pathways were interrelated and shared common nodes with other 206 metabolic pathways (e.g., bile acid biosynthesis, galactose glycerophospholipid, 207 glycosphingolipid, and linoleate metabolism). In addition, we utilized metabolite set enrichment 208 analysis (MSEA), which takes into account the quantitative measurement of each metabolite and 209 groups them into functionally related sets from a collection of predefined human metabolic 210 pathways (Jia et al., 2014) . Results from MSEA were consistent with observations from network 211 reconstruction, suggesting a prominent role for lipids upon early BCG vaccination in newborns 212 ( Figure 3C , Table S3 ). Early BCG administration was associated with increased production of 213 multiple metabolites of the glycosphingolipid and glycerophospholipids, including LPCs. In 214 contrast, glycerol, phosphoethanolamines, and cortisol were decreased in the early vs. delayed 215 BCG group. 216 217 To gain further insight into the effects of BCG on the infant plasma lipidome, we assayed 218 the same in vivo samples (i.e., those characterized using the untargeted metabolomics platform) 219 using Complex Lipid Panel LC-MS/MS-based lipidomics ( Figure 4A ). Across the 14 lipid 220 classes identified by this assay, we identified a total of 963 lipids, with triacylglycerol (TAG) 221 being the most abundant lipid subclass, followed by phosphatidylethanolamines (PEs) and 222 phosphatidylcholine ( Figure 4B ). Separation of groups by the timing of BCG vaccination (early 223 vs. delayed) accounted for 52% variance at Comp 1 and 15% at Comp 2 ( Figure 4C ). A total of 224 30 lipids (3.1%) were significantly perturbed, comparing early vs. delayed BCG newborns 225 ( Figure 4D ) (Table S4) . 233 234 in vivo 236 237 We hypothesized that BCG-induced metabolic signatures detected in vivo could be 238 modeled in vitro. To this end, we obtained human cord blood from a Boston newborn cohort 239 (full-term newborns delivered via cesarean section) and assessed responses utilizing a whole 240 blood assay (WBA) ( Figure 1B 437 metabolites after quality control and assurance and filtering out the xenobiotics to focus on 249 endogenous metabolites ( Figure S2F -J). BCG-only samples (i.e., no blood) at low and high 250 dilution were also assayed as additional controls to assess background and identify metabolites 251 produced by the BCG vaccine itself ( Figure S3A ). BCG-only controls demonstrated lower 252 measurable concentrations in most lipid families ( Figure S3B ). Also, they had a lower number of 253 detected lipids ( Figure S3C ) compared to cord blood stimulated with vehicle or with BCG. 254 Matched pairs or pairwise comparisons of samples from the same participant were used for 255 analysis. 256 257 Principal component analysis (PCA) of metabolites detected in plasma from the in vitro 258 stimulation assay illustrated that BCG treatment induced changes in the plasma metabolome 259 ( Figure 5A ). Hierarchical clustering demonstrated the grouping of metabolite levels by treatment 260 ( Figure 5B ). Metabolite set enrichment analysis (MSEA) using relative metabolite 261 concentrations from the Boston in vitro cohort identified the glucose-alanine cycle, lactose 262 degradation pathway, and sphingolipid metabolism as the top enriched pathways after BCG 263 stimulation ( Figure 5C ). Metabolites that significantly differed between BCG-stimulated vs. 264 control conditions in vitro included 25 upregulated metabolites and 77 down-regulated 265 metabolites ( Figure 5D , Table S5 ). A comparison between the in vivo Guinea Bissau and the in 266 vitro Boston cohorts revealed 47 shared enriched metabolic pathways, representing a 58% 267 overlap ( Figure 7A ). The number of unique pathways identified in BCG-stimulated samples in 268 vitro (19 pathways: 23.5%) was slightly higher than that in the early BCG group in vivo (15 269 pathways: 18.5%) ( Figure 7A ). Our results suggest more acute metabolic changes detected in 270 vitro (18 hr post-BCG stimulation) than four weeks post in vivo BCG vaccination. 271 272 Lipidomic analysis of supernatants from whole blood stimulated with BCG in vitro 273 identified 963 lipids belonging to 15 families. Among those, only 75 lipids (7.6%) were 274 significantly perturbed, with most lipids decreased upon BCG stimulation ( Figure 4E ). BCG 275 stimulation induced a decrease of several plasma phospholipids metabolism intermediates, such 276 as choline phosphate, glycerophosphocholine (GPC), and phosphoethanolamine (PE) (Table S7) . 277 Similar to total metabolic changes, more acute differences were noted for lipids stimulated in 278 vitro (18 hr stim) compared to in vivo (4 weeks post-vaccine), possibly reflecting differences in 279 the kinetics of BCG-induced lipid changes ( Figure 4F ). Multiple LPC components (LPC 16:0, 280 18:2, 20:4, 22:6) and other lipids were also decreased in BCG-stimulated samples in vitro 281 (Figure 4G). 282 283 Previous studies have assessed the role of fatty acids and the eicosanoid pathway in Mtb 284 infection and BCG vaccine efficacy (McFarland et al., 2008) . Eicosanoids are a family of lipid 285 mediators involved in inflammation ( Figure S3F ). Prostaglandin E 2 (PGE 2 ) was significantly 286 increased upon BCG stimulation ( Figure S3D ), while we observed a significant reduction in 287 docosahexaenoic acid (DHA 22:6 (n-3)) ( Figure S3E ) in BCG-stimulated samples vs. control. 288 Linoleic acid (FFA 18:2) and arachidonic acid (FFA (20:4)), an immediate precursor of PGE 2 289 ( Figure S3G -H), were both decreased in BCG-stimulated samples compared to control. PGs were 290 not detected in the Guinea-Bissau in vivo cohort (Table S2) The immunogenicity of BCG in the Guinea-Bissau in vivo cohort, as measured by whole 307 blood cytokine responses to PPD antigen and TLR-agonist stimulation, has been previously 308 reported (Jensen et al., 2015) . To study the relationship between BCG-induced LPLs in vivo 309 and recall cytokine production in vitro, we analyzed previously generated cytokine 310 measurements on PPD and other TLR agonist recall responses for the subset of newborn 311 participants included in our metabolomic and lipidomic studies. Relative to the delayed BCG 312 cohort, newborns who received early BCG demonstrated increased PPD-induced production of 313 TNFα (p=0.0072), IL5 (p<0.0001), IL6 (p=0.0229), IL17 (p<0.0001), and IFNγ (p<0.0001) 314 ( Figure S4A -G, To determine the response to stimulation with BCG in vitro, we subjected WBA 326 supernatants to multiplex cytokine and chemokine profiling (Figures S1B and 1B). BCG 327 stimulation of cord blood for 18 hr significantly induced the production of multiple cytokines 328 and chemokines, resulting in a balanced Th1-, Th2-, and Th17-polarizing cytokine profile 329 (Figure S5A). Consistent with previously reported acute activation of BCG-induced Th1 330 responses (Angelidou et al., 2020a) and early proinflammatory bias (Freyne et al., 2018) , the 331 addition of BCG to whole blood in vitro induced IL-1ß, a cytokine that may be important to 332 BCG vaccine immunogenicity (Scheid et al., 2018) and innate training that may contribute to 333 non-specific/pathogen-agnostic beneficial effects (Arts et al., 2018) ( Figure S5B ). BCG also 334 induced the production of anti-inflammatory IL-10 ( Figure S5B ). As expected, these plasma 335 cytokines were not detected in vivo at the 4-week time point, given likely normalization to 336 baseline 4-weeks after vaccination (Stenken and Poschenrieder, 2015) . 337 338 Comparison of BCG-induced metabolic profiles across cohorts demonstrates modulation of 339 common metabolomic pathways 340 341 To validate metabolomic signatures of the BCG vaccine in early life, we compared the 342 Guinea-Bissau and Boston cohorts with another independent newborn group from the EPIC-001 343 study in The Gambia (West Africa) using high-throughput metabolomics. Newborns (n=27) were 344 assigned to either receive the Expanded Program on Immunization (EPI) vaccines (OPV, BCG, 345 and HBV) at birth or delayed during the first week of life to study the effect of vaccine responses 346 on early life immune ontogeny ( Figure 1C , Figure S1C ) (Lee et al., 2019) . Peripheral blood was 347 collected at two time points: a pre-vaccination sample at the day of life 0 (DOL0; birth), then a 348 second sample randomized at either DOL1, DOL3, DOL7. All enriched metabolite sets 349 discovered in the EPI-vaccinated Gambia cohort were shared among the Guinea-Bissau and 350 Boston cohorts suggesting that BCG-induced metabolic pathways followed a consistent pattern 351 ( Figure 7A , 7C, 7D). Shared sets of BCG-induced metabolic changes included sphingolipid 352 metabolism, glycine and serine metabolism, methionine metabolism, purine metabolism, 353 galactose metabolism, bile acid biosynthesis, amino sugar metabolism, lactose synthesis, 354 spermidine and spermine biosynthesis, and betaine metabolism ( Figure 7D ). Figure 7B ). Of 361 note, ~70% of significant metabolites in Gambian neonates belonged to the lipid class (Table 362 S8), supporting a major lipid signature of BCG vaccination early in life. 363 364 365 366 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted December 5, 2021. In our study, BCG-induced changes in LPCs correlated with blood cytokine responses to 428 stimulation with multiple TLR agonists and mycobacterial antigen (PPD) ( Figure 6G -6H). 429 Interestingly, potential pro-inflammatory effects of BCG vaccination may be counterbalanced by 430 enhanced production of LPC 16:0, an LPC that was increased in our in vivo cohort ( Figure 6A ). LPC 16:0 can act as an "eat-me" signaling target, enhancing uptake of apoptotic cells ( . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted December 5, 2021. ; Upon stimulation of human newborn blood with BCG in vitro, we noted a significant increase in 459 PGE 2 and arachidonate acid (FFA (20:4)), an immediate precursor of prostaglandin E 2 (PGE 2 ). 460 Possibly specific and -agnostic protection should adopt study designs amenable to head-to-head strain 494 comparisons in order to detect actionable differences in vaccine efficacy and immune signatures 495 (Comstock, 1988 ). 496 497 Our study features a number of strengths, including (a) a focus on human immune 498 responses to BCG, one of the most commonly given vaccines across the globe that is critical to 499 newborn health and is also being studied for potential benefits in protecting against viral . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. ; identification of lipids and in particular LPLs/LPCs as key metabolites altered by BCG; (e) 505 significant correlation of LPCs with innate (TLR agonist) and adaptive (PPD) cytokine 506 responses; and (f) validation of BCG-induced metabolic signatures across three independent 507 cohorts in West Africa (Guinea Bissau and The Gambia; in vivo) and North America (Boston, 508 USA; in vitro). Overall, by demonstrating the applicability of metabolomics in vulnerable 509 newborns from resource-poor settings and defining novel candidate biomarkers that may 510 contribute to BCG's protective effects, our study represents an important advance in the field of 511 neonatal systems vaccinology. 512 513 Our study also has several limitations, including (a) limited blood sample volumes from 514 newborns necessitating pooling of samples by sex and treatment prior to metabolomic and 515 lipidomic analyses, (b) a relatively high neonatal mortality in the early BCG trial so that some 516 selection bias may have occurred, as we were only able to assess those who survived to 4 weeks 517 of age, and (c) a possibility for false-positive findings for the Guinea-Bissau samples, as we 518 chose not to adjust for multiple comparisons (Rothman, 1990) . Regarding the latter, many false 519 discovery rate (FDR) methods are considered too stringent for metabolomics analysis due to the 520 high correlation and redundancy between metabolite features, resulting in a lack of agreed-upon 521 standards in the field. We mitigated FDR concerns by validating our findings using a human 522 newborn in vitro system and cross-comparison with a second independent Gambian newborn 523 cohort studied in vivo to model BCG-induced metabolic shifts. Finally, BCG may induce a wider 524 variety of metabolic pathways, and future studies are required to identify their significance and 525 functional relation to immunogenicity and protection, as well as their correlation with other 526 systems biology ("omic") measures such as systems serology (Ackerman et al., 2017) . 527 528 In summary, we have demonstrated the feasibility of assessing neonatal plasma 529 metabolites in a resource-poor setting to identify vaccine-induced metabolic pathways in this 530 study. BCG-induced alterations in LPLs, especially LPCs known to have roles in immune 531 regulation and response to infection (Carneiro et al., 2013), were particularly pronounced and 532 correlated with innate (TLR agonist) and adaptive (PPD) cytokine responses, suggesting that 533 LPCs may contribute to BCG immunogenicity. These lipid biomarkers are novel candidates for 534 correlates of protection that may contribute to BCG's specific (vs. TB) and heterologous 535 protective effects. Overall, our study suggests that vaccine-induced metabolites, and especially 536 lipids, may be relevant biomarkers of vaccine immunogenicity that may help inform more 537 precise discovery and development of vaccines. 538 539 540 541 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. Requests for information regarding reagents and resources should be directed to and will be 565 fulfilled by correspondence to Dr. assigned to the control group were treated according to local practice such that vaccination was 593 postponed until they reached >2.5 kg in weight, or more commonly when they presented for their 594 first Penta vaccine (DTP-HBV-Hib) recommended at six weeks of age. The Pentavalent vaccine 595 given to infants was either Easyfive (Panacea Biotech India), Quinvaxem (Berna Biotech Korea 596 Corp) or Pentavac (Serum Institute of India) for this randomized control study. All infants 597 received oral polio vaccine (OPV) at birth. For enrollment into the immunological sub-group 598 study, infants were visited at home by the study team four weeks after randomization to BCG or 599 no BCG. For logistical reasons, infants living in Bissau City and nearby suburbs were given 600 priority. After providing informed consent, the mother was interviewed about the health status of 601 her child, and the length, weight, and mid-upper arm circumference of the child were measured. 602 Clinical data on sex, weight, BCG scar, PPD testing results were also available. Scar size was 603 defined as the average of two perpendicular diameters on the scar formed at the injection site. 604 Local reaction to the BCG vaccine was assessed and measured. Blood was collected by heel 605 puncture into a heparin-coated tube. 606 607 For this current study, we included plasma samples from newborns randomized within 608 the first week of life. However, samples from infants in the delayed BCG group who had 609 received BCG before phlebotomy, who were Penta-vaccinated, or had hemolyzed samples were 610 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. ; excluded. Biosamples were shipped on dry ice to the Precision Vaccines Program (Boston, MA, 611 USA) for subsequent metabolomic assays ( Figure S1 ). In vitro cytokine assays were conducted 612 as outlined below. For metabolomic and lipidomic assays, samples were selected per sex and 613 treatment based on the following criteria: 1) Exclusion criteria included a history of vomiting or 614 diarrhea per mother's report, hemolyzed samples, or limitation of available sample volume (i.e., 615 <20µL); 2) Inclusion: To overcome the limitation of sample volumes, ten newborn samples 616 stratified by sex and treatment were pooled prior to shipment to Metabolon (Durham, North 617 Carolina, USA) for mass spectrometry-based metabolomics and lipidomic assays. These samples 618 were matched with their clinical information. To gain insight into the impact of BCG 619 immunization on both innate and adaptive immunity, blood was collected at four weeks and 620 stimulated in vitro with vehicle control (Roswell Park Memorial Institute medium, RPMI), Toll-621 like receptor (TLR) agonists or PPD. After incubation, supernatants were collected and 622 cryopreserved prior to batch measurement of cytokines by multiplex assay. Clinical data with 623 corresponding in vitro stimulated blood derived from the early BCG (n=60) and control infants 624 (delayed BCG, n=60) included in this metabolomics study were reanalyzed and yielded 625 comparable baseline clinical characteristics ( Hepatitis B) at birth or after a delay. A follow-up blood sample collection was obtained from all 649 infants at either DOL 1, DOL 3, or DOL 7 with a maximum of two peripheral blood collections 650 per participant in the first week of life ( Figure 1C , Figure S1C ). Plasma preparation and 651 metabolomics assay for this cohort have been previously described ( To enable an 658 association with the original source identifier and for tracking purposes, each sample was 659 assigned a LIMS unique identifier. Samples were extracted and prepared for analysis using 660 Metabolon's solvent extraction method (Evans, 2008) . Recovery standards were added to the first 661 step in the extraction process to ensure proper quality control. Protein was removed by methanol 37°C humidified incubator with 5% CO 2 for 24 hr to assess the mycobacterial specific response 703 together with RPMI control. After 24h of culture, supernatants were collected and stored at < 704 −70°C until analysis. Cytokine concentrations were measured at SSI, employing an 705 immunobead-based multiplexed assay as previously described (Skogstrand et al., 2005) . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. Tables S9 and S10. 781 782 Targeted Assays 783 Plasma cytokine and chemokine concentrations were measured by multiplex assay, using 784 a Flexmap 3D system with Luminex xPONENT software version 4.2 (Luminex Corp.; Austin, 785 TX, USA). Cytokines and chemokines were measured using Milliplex Analyst software (v. 786 3.5.5.0, Millipore). Infant blood from the Guinea Bissau infant cohort was diluted and stimulated 787 in vitro with PPD or TLR agonists and cytokine and chemokine production measured as 788 previously described (Jensen et al., 2015) . Cytokine and chemokine production from blood 789 derived from the subset of newborn participants included in our metabolomics and lipidomics 790 study was analyzed using an unpaired Wilcoxon Rank-sum Test. Comparisons with p<0.05 were 791 considered statistically significant with figure asterisks denoting level of significance (*p<0.05; 792 **p<0.01; ***p<0.001). Data are depicted as medians. 793 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. ; presented are the scaled, normalized peak intensities of the metabolites. * P < 0.05; 920 ** P < 0.01; *** P < 0.001 vs. control. between early and delayed BCG groups with PC1 accounting for 52% of variance and 940 PC2 for 15% variance. 941 (D) Early administration of BCG at birth significantly perturbs plasma lipid concentrations at 942 4 weeks of age. Significantly altered lipid metabolites in the early vs. delayed BCG 943 newborn group were annotated in red (P < 0.05), those that were nearly significant in 944 pink (P < 0.1 to P > 0.05), and those that were not significant in gray. 945 (E) Stimulation of human newborn cord blood in vitro with BCG perturbs phospholipid 946 pathways. Supernatants from blood stimulated in vitro for 18 hr with vehicle control, or 947 BCG were subjected to lipidomics and demonstrated much lower production of lipids 948 compared to control. Significantly altered lipid metabolites in the BCG vs. vehicle 949 conditions were annotated in red (P < 0.05), those that were nearly significant in pink (P 950 < 0.1 to P > 0.05), and those that were not significant in gray. MetaboAnalyst) highlighted pathways that were prominently altered after BCG 975 stimulation, including those relating to the glucose-alanine cycle, lactose, and 976 sphingolipid metabolism. Fold enrichment was calculated by dividing the observed 977 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. ; number of hits by the expected number of hits of the overrepresented pathway. MSEA 978 calculates a hypergeometric test score employing a cumulative binomial distribution 979 based on the probability of seeing at least a particular number of metabolites with the 980 biological term of interest in a given compound list. 981 (D) The volcano plot illustrated BCG-induced changes in metabolites as compared to vehicle 982 control. Red color represents significant (P < 0.05), pink marginally significant (P < 0.1 983 to P > 0.05), and gray non-significant lipids. 984 985 Figure 6 : BCG-induced plasma lysophospholipids correlated with TLR-and mycobacterial 986 antigen-induced cytokine responses. 987 Peripheral blood was collected from early vs. delayed BCG immunized infants in Guinea Bissau 988 at 4 weeks of age and diluted for in vitro TLR agonist-and PPD-induced stimulation assay as 989 previously described (Jensen et al., 2015) . is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. ; https://doi.org/10.1101/2021.12.03.21267251 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. ; . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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decade after the metabolomics standards 1410 initiative it's time for a revision Bioanalytical chemistry of cytokines--a review Proposed minimum reporting standards for 1415 chemical analysis Chemical Analysis Working Group (CAWG) Metabolomics Standards 1416 Initiative (MSI) Out-of-sequence DTP and 1418 measles vaccinations and child mortality in Guinea-Bissau: a reanalysis Phospholipid Species in Newborn and 4 Month Old Infants after Consumption of Different 1421 Formulas or Breast Milk Fewer out-of-sequence vaccinations and reduction of child mortality in Northern 1424 A 1426 genomics-based approach to assessment of vaccine safety and immunogenicity in children Neonatal Immunization: 1429 Rationale, Current State, and Future Prospects Children: Improving Survival and Well-being MetaboAnalyst 3.0--making 1432 metabolomics more meaningful Metabolomic data processing, analysis, and interpretation 1434 using MetaboAnalyst Using MetaboAnalyst 3.0 for Comprehensive Metabolomics 1436 Data Analysis Therapeutic effects of lysophosphatidylcholine in experimental 1439 sepsis 1441 van der Klis 1444 1445 1446 RE2717 R01194 RE2717 R01184 R02843 RE2763 R02842 R02840 R02838 5(S)-HETE 5(S)-HPETE "15(R)-Hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoate early BCG group demonstrated significantly greater PPD-induced production of TNFα, IL5, 1079 IL6, IL17 and IFNγ. Groups were compared using the Wilcoxon Rank Sum Test. Data are 1080 depicted as geometric means. * P < 0.05: ** P < 0.01; *** P < 0.001 1081 1082 Figure S5 : BCG-induced cytokine and chemokine production in Boston human newborn 1083 cord blood in vitro. (A) Heatmap depicting changes in the production of 41 selected cytokines and chemokines 1085 after BCG in vitro stimulation for 18 hr. Two-sample t-tests with Benjamini, Krieger and 1086Yekutieli correction comparing BCG-stimulated vs. vehicle control * P < 0.05: ** P < 1087 0.01; *** P < 0.001. Data are reported using Euclidean distance and Ward algorithm. 1088(B-E) Selected inflammatory cytokines showed differences between vehicle control 1089 stimulated cord blood vs. BCG-stimulated cord blood. Paired student t-test used for data 1090 analysis * P < 0.05: ** P < 0.01; *** P < 0.