Trans-vaccenic acid (TVA) and derivatives thereof for enhancing immune development and responses in newborns

Abstract

Provided herein are compositions and methods for enhancing immune development and responses in newborns. In particular embodiments, pre-natal and post-natal supplements and or infant formula and or follow-on formula (FOF) are provided comprising long chain fatty acids (LCFAs) and very long chain fatty acids (VLCFAs), such as trans-vaccenic acid (TVA), cis-11,14-Eicosadienoic acid (EDA), and nervonic acid (NVA), or derivatives thereof.

Description

[0001] Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0002] TRANS-VACCENIC ACID (TVA) AND DERIVATIVES THEREOF FOR ENHANCING IMMUNE DEVELOPMENT AND RESPONSES IN NEWBORNS

[0003] CROSS-REFERENCE

[0004] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 682,199, filed August 12, 2024; which is incorporated by reference herein in its entirety.

[0005] FIELD

[0006] Provided herein are compositions and methods for enhancing immune development and responses in newborns. In particular embodiments, pre-natal and post-natal supplements and or infant formula and follow-on formula (FOF) are provided comprising long chain fatty acids (LCFAs) and very long chain fatty acids (VLCFAs), such as trans-vaccenic acid (TVA), cis- 11,14-Eicosadienoic acid (EDA), and nervonic acid (NVA), or derivatives thereof.

[0007] BACKGROUND

[0008] Newborns and infants are more susceptible to infections than the general population. Babies born prematurely are more likely to get infections that can lead to sepsis and bronchopulmonary dysplasia (BPD). This is because they have a weaker immune system. Infection can happen through equipment such as tubes into a vein or ventilation tubes, which help support the preterm baby’s breathing. Preterm infants have a lower number of immune cells, and lower functional capacity compared with term infants. Moreover, preterm infants fed breast milk have lower infection-related disease rates compared with preterm infants with formula. Identifying components of breast milk that are important for premature infants' immune system development is of critical importance.

[0009] SUMMARY

[0010] Provided herein are compositions and methods for enhancing immune development and responses in newborns. In particular embodiments, pre-natal and post-natal supplements and or infant formula and follow-on formula (FOF) are provided comprising long chain fatty acids (LCFAs) and very long chain fatty acids (VLCFAs), such as trans-vaccenic acid (TVA), cis- 11,14-Eicosadienoic acid (EDA), and nervonic acid (NVA), or derivatives thereof. Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0011] In some embodiments, provided herein are prenatal and / or postnatal supplement comprising trans-vaccenic acid (TV A), cis-l l,14-Eicosadienoic acid (EDA), and / or nervonic acid (NVA). In some embodiments, the supplement further comprises one or more of folate, calcium vitamin A, riboflavin, iodine, DHA, omega-3 fatty acids (e.g., docosahexaenoic acid (DHA)), pyridoxine, iron, choline, niacin, thiamin, magnesium, selenium, vitamin D, vitamin C, vitamin B 12, vitamin E, and zinc. In some embodiments, the supplement is formulated for oral administration to a pregnant or lactating subject. In some embodiments, the supplement comprises TVA. In some embodiments, the supplement comprises EDA. In some embodiments, the supplement comprises NVA.

[0012] In some embodiments, provided herein are methods of enriching breast milk with TVA, EDA, and / or NVA comprising administering a prenatal and / or postnatal supplement of claim 1 to a pregnant or nursing subject.

[0013] In some embodiments, provided herein are methods of enhancing immune development and / or immune response in a newborn or infant or pediatric subject comprising administering breast milk or infant formula or follow-on formula (FOF) comprising TVA, EDA, and / or NVA to the subject.

[0014] In some embodiments provided herein are methods of enhancing immune development and / or immune response in a newborn or infant or pediatric subject comprising (a) administering a supplement comprising TVA, EDA, and / or NVA to a pregnant or lactating subject, and (b) feeding the newborn or infant or pediatric subject with breast milk from the pregnant or lactating subject.

[0015] In some embodiments, provided herein are infant or baby formulas comprising trans- vaccenic acid (TVA), cis-l l,14-Eicosadienoic acid (EDA), and / or nervonic acid (NVA). In some embodiments, infant or baby formulas further comprise whey, casein, whey protein isolate, skim milk powder, vegetable oils, animal milk fats, milk oligosaccharides, iron, docosahexaenoic acid (DHA), arachidonic acid (ARA), folate, amino acids, and / or taurine. In some embodiments, the formula is formulated for oral administration to a newborn or infant or pediatric subject. In some embodiments, the supplement comprises TVA. In some embodiments, the supplement comprises EDA. In some embodiments, the supplement comprises NVA.

[0016] In some embodiments, provided herein is the use of an effective dose of trans-vaccenic acid (TVA), cis-11,14-Eicosadienoic acid (EDA), and / or nervonic acid (NVA) for enhancing Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 immune development and / or immune response in a newborn or infant or pediatric subject.

[0017] In some embodiments, provided herein is the use of an effective dose of trans-vaccenic acid (TVA), cis-11,14-Eicosadienoic acid (EDA), and / or nervonic acid (NVA) in the manufacture of a supplement for administration to a pregnant or lactating subject.

[0018] In some embodiments, provided herein is the use of an effective dose of trans-vaccenic acid (TVA), cis-11,14-Eicosadienoic acid (EDA), and / or nervonic acid (NVA) in the manufacture of a infant or baby formula for administration to a newborn or infant or pediatric subject.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1. TVA enhances neonatal T cell function.

[0021] Figure 2. TVA-enriched breast milk advances T cell immunity against viral infection in newborn mice.

[0022] Figure 3. 11 preterm infant blood spot card samples with paired diet (milk or formula) samples. Fatty acid profile of the diet samples was compared the FA levels between infants with infection-related diseases (sepsis and BPD) and no disease. Higher intake of TVA in preterm infants reduced the incidence of sepsis and BPD.

[0023] Figure 4. The neonatal immune system differs significantly from that of adults. To explore this, we conducted ex-vivo screens to identify the specific roles of milk fatty acids in regulating neonatal immune cells compared to adult immune cells. Our findings indicate that TVA treatment enhances the function of neonatal T cells more effectively than it does for adult T cells. We plan to test additional immune cells in future studies.

[0024] Figure 5. including CD8+ and CD4+ T cells by assessing by IL-2, TNFa, and IFNy production levels (left two panels, respectively), and natural killer (NK) cells and dendritic cells (DCs) (right two panels, respectively).

[0025] Figure 6. Exemplary NVA derivatives.

[0026] Figure 7. Exemplary EDA derivatives.

[0027] Figure 8. Exemplary TVA derivatives.

[0028] Figure 9A-H. Maternal TVA reshapes neonatal T cell development via breastfeeding. (A) Analysis results of correlation between TVA levels in human breast milk from mothers / donors and serum samples on blood spot cards from their preterm babies (n=l 1 samples). The schematic Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 figure was generated using BioRender. (B) Schematic depicting the experimental design of feeding mouse breeders with TV A or CON diets to check their offspring’s immune development. The figure was generated using BioRender. (C) TVA levels in breast milk from breeders on TVA- enriched diet when their pups are at 1 or 2 weeks old (Iw: n=3 CON, n=3 0.1%TVA, n=4 1% TVA; 2w: n=3 CON, n=40.1%TVA, n=4 1% TVA). (D) TVA levels in serum samples of mouse pups at 1, 2, 3, or 5 weeks old (Iw: n=3 CON, n=l 0.1%TVA, n=3 1% TVA; 2w: n=3 CON, n=3 0.1%TVA, n=2 1% TVA; 3w: n=3 CON, n=20.1%TVA, n=4 1% TVA; 5w: n=3 CON, n=3 0.1%TVA, n-5 1% TVA). (E) Effects of TVA-milk on pups’ spleen weight (Iw: n=4 CON, n=4 0.1%TVA, n=14 1% TVA; 2w: n=8 CON, n=8 0.1%TVA, n=3 1% TVA; 3w: n=14 CON, n=5 0.1%TVA, n=5 1% TVA; 5w: n=ll CON, n=8 0.1%TVA, n=8 1% TVA). (F) TVA-milk’s effects on single-nuclei multi-omics (snRNA-seq & snATAC-seq) of 2-week-old pups’ splenic cells. UMAP plot displaying different subsets from single-nuclei multi-omics. (Five mice were pooled per group). (G) Cell proportion changes (TVA vs. CON) of different subsets in 2-week-old pups’ splenic cells. (H) Gene promoter sum (snATAC-seq based) changes between TVA and CON group in 2-week-old pups’ splenic T cells. (I) Each cell cluster’s Gpr43 Sum changes between TVA and CON group in 2-week-old pups’ splenic cells. The correlation coefficient (r) and statistical significance (P-value) were determined using Pearson correlation analysis, the shaded area represents the 95% confidence interval (A). Data are mean (C,D) or mean ± s.e.m. (E). Statistical analysis was performed using two-way ANOVA (E).

[0029] Figure 10A-F. TVA-milk reprograms neonatal naive CD4+T cells through a GPR43- CTCF axis. (A) Schematic depicting the design of ex-vivo experiments to examine neonatal naive CD4+T cells from the offspring of C57BL / 6 or Gpr43 knockout mouse breeders fed on either a control (CON) or TVA diet (left). Following naive CD4+T cell activation or stimulation, changes in Thl polarization rate, percentage of CD69+cells, and cell expansion are shown (right) (n=3 each). The schematic figure was generated using BioRender. (B) TVA-milk’s effects on naive CD4+T cell subsets (from Cluster 3 in figure If) in 2-week-old pups’ splenic cells. (C) Cell number changes (TVA vs. CON) of different naive CD4+T cell subsets in 2-week-old pups’ splenic cells. (D) The motif score of CTCF in different naive CD4+T cell subsets in 2-week-old pups’ splenic cells. (E) Changes in Thl polarization rate (n=5 each) and TBX21 (T-bet) activity (n=3 each) following in vitro treatment of neonatal naive CD4+T cells with TVA or 8-Bromo- cAMP, under siNTC, siCtcf, or siCrebl conditions. (F) Changes in CTCF activity following in Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 vitro treatment of neonatal naive CD4+T cells with TVA, with or without 8-Bromo-cAMP or PKA inhibitor (n=3 each). Data are mean ± s.d. (A, E, F). Statistical analysis was performed using two- way ANOVA (A, E, F).

[0030] Figure 11 A-J. TVA-milk primes the readiness of neonatal T cells against lung viral infection. (A) Schematic depicting the experimental design of influenza A virus challenge for neonatal mice (10-day-old) with / without TVA in breastmilk. The figure was generated using BioRender. (B) Effects of TVA in breast milk on the survival rate of mice with infection by influenza A virus at 10-day-old (n=17 CON, n- 14 TVA). (C) Effects TVA-milk on lung epithelial damage (assessed by Keratin 5 staining) and normal epithelium (assessed by pro- surfactant protein C staining) of 10-day-old mouse pups at 7 days post-viral infection in lungs. Data summaries are shown (no virus: n=4 CON, n=5 TVA; with virus: n=6 each). (D) TVA-milk enhanced anti-viral immunity of pups with reduced viral loads in lungs assessed by decreased influenza virus PR8 NS1 mRNA levels on Day 7 and 10 post-infection to pups at 10-day-old (D4: n=8 CON, n=7 TVA; D7: n=7 CON, n=6 TVA; DIO: n=6 CON, n=7 TVA). (E) Effects of TVA-milk on lung-infiltrating neonatal CD4+T cell activation assessed by CD69 (left) and virus-recognizing activity assessed by peptide NP331-325 (viral nucleocapsid (N) protein)- specific cells (right) (D4: n=8 CON, n=7 TVA; D7: n=7 CON, n=7 TVA; DIO: n=6 CON, n=7 TVA; n=4 no virus). (F) Effects of TVA-milk on lung-infiltrating neonatal CD8+T cell activation (CD69, left) and virus-recognizing activity (PA224- 233-specific, right) (D4: n=8 CON, n=7 TVA; D7: n=7 CON, n=7 TVA; DIO: n=6 CON, n=7 TVA; n=4 no virus). (G) Effects of TVA-milk on lung ZL-1 level of 10-day-old mouse pups at 4, 7, andlO days post-viral infection (n=5 each). (H) SnRNA-seq analysis of lung tissue cells of pups with influenza A viral infection. t-SNE plot displaying different lung cell subsets from snRNA-seq (samples from 5 mice / group pooled). (I) Cell proportion changes (TVA vs. CON) of different subsets in H. (J) Schematic depicting the experimental design of influenza A virus challenge for neonatal Tbx2Tl~ mice (10-day-old) with / without TVA in breastmilk. The figure was generated using BioRender (left). Effects of TVA in breast milk on the survival rate of Tbx2 '~ mice with lung infection by influenza A virus at 10-day-old (right) (n=4 CON, n=8 TVA). Data are Kaplan- Meier overall survival curves (B, J), mean ± s.d. (C-F), or mean± s.e.m. (G). Statistical analysis was performed using log-rank (Mantel-Cox) test (B, J) or two-way ANOVA (C-G).

[0031] Figure 12A-H. TVA-milk inversely correlates with incidence of human preterm BPD and reprograms mouse neonatal naive CD4 T cells with long-lasting imprinting. (A) Analysis results Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 of correlations between levels of TVA and cytokines including IL-2 and IL-4 in preterm infant serum samples (n = 11 samples). The schematic figure was generated using BioRender. (B) Fatty acid profiling analysis results are shown, indicating that TVA levels in breast milk samples from human mothers or donors inversely correlate with the chance for then- preterm babies to have BPD (no BPD n=16, BPD n=6). The schematic figure was generated using BioRender. (C) TVA levels in serum samples from preterm infants inversely correlate with their incidence of BPD (no BPD n=8, BPD n=3). (D) Schematic depicting the experimental design to examine TVA-milk’s effects on immune imprinting. (E) TVA-milk’s effects on serum TVA concentration of offspring at wean (3 weeks old) or adulthood (6-10 weeks old) (n=3 each). (F) Effects of having TVA enriched breast milk before wean (0-3 weeks old) on the survival rate with infection by influenza A virus at 6-10 weeks old (n=9 CON to CON, n=7 TVA to CON). (G) Differences in CTCF activity in naive CD41T cells from adult mice that were fed with TVA-enriched or control milk during the preweaning period (0-3 weeks of age) (n=3 each). (H) Differences in Thl polarization rates of naive CD4+T cells upon activation in adult mice that were fed TVA-enriched or control milk during the pre-weaning period (0-3 weeks of age) (n=3 each). The correlation coefficient (r) and statistical significance (P-value) were determined using Pearson correlation analysis, the shaded area represents the 95% confidence interval (A). Data are mean ± s.d. (C, G, H), mean (E), or Kaplan- Meier overall survival curves (F). Statistical analysis was performed using Student’s two-sided unpaired t-test (B, C, G, H), or log-rank (Mantel-Cox) test (F).

[0032] Figure 13A-M. TVA-milk’s effects on the immune system of mouse pups. (A) Effects of TVA-milk on pups’ body weight. (B) TVA-milk’s effects on immune cell number of 2-week-old pups’ spleens (splenocyte, CD4+, CD8+, B: n=14 each; neutrophil, DC, monocyte: n=8 each). (C) TVA-milk’s effects on immune cell percentage of splenocytes of 2-week-old pups’ spleens (splenocyte, CD4+, CD8+, B: n=14 each; neutrophil, DC, monocyte: n=8 each). (D) TVA-milk’s effects on immune cell number of 5-week-old pups’ spleens (n=ll CON, n=8 TVA). () TVA- milk’s effects on immune cell percentage of splenocytes of 5-week-old pups’ spleens (n=l 1 CON, n=8 TVA). (F-G) Effects of TVA-milk on pups’ splenic CD4+(F) or CD8+(G) T cell numbers at 1, 2, 3, and 5 weeks old (Iw: n=4 CON, n=4 TVA; 2w: n=14 CON, n=14 TVA; 3w: n=5 CON, n=6 TVA; 5w: n=l 1 CON, n=8 TVA). (H) Effects of TVA-milk on pups’ spleen naive, central memory, and effector memory CD4+T cells at 2 weeks old (n=14 each). (I) Effects of TVA-milk on pups’ splenic Treg cells at 2 weeks old (n=8 CON, n=3 TVA). (J) Effects of TVA-milk on Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 pups’ splenic naive, central memory, and effector memory CD8+T cells at 2 weeks old (n=14 each). (K) Effects of TVA-milk on pups’ splenic %CD69+of CD4+or CD8+T cells at 2 weeks old (n=14 each). (L) Effects of TVA-milk on pups’ thymus weight at 2 weeks old (n=16 CON, n=19 TVA). (M) Effects of TVA-milk on the thymic development of pups at two weeks old, including the percentages of double-negative (DN), double-positive (DP), CD4 single-positive (4SP), and CD8 single-positive (8SP) thymocytes (n=14 each). Data are mean ± s.d. (A-M). Statistical analysis was performed using two-way ANOVA (A, F-G) or Student’s two-sided unpaired t-test (B-E, H-M).

[0033] Figure 14A-C. The effects of TVA on the neonatal spleen immune cells. (A) Each cell cluster’s CREB1_MAOO18.4 motif changes between TVA and CON group in 2-week-old pups’ splenic cells. (B) Each cell cluster’s TBX21_MA0690.1 motif changes between TVA and CON group in 2-week-old pups’ splenic cells. (C) Ex-vivo experiments to examine neonatal naive CD41T cells from the offspring of C57BL / 6 or Gpr43 knockout mouse breeders fed on either a control (CON) or TVA diet. Following activation or stimulation, changes in Th2 and Thl7 polarization rate are shown (n=3 each). Data arc mean ± s.d. (C). Statistical analysis was performed using two- way ANOVA (C).

[0034] Figure 15A-G. The effects of TVA on the neonatal naive CD4+T cells are mediated through a GPR43-cAMP-PKA-CTCF axis. (A) Schematic depicting the design of in-vitro experiments to examine the effects of TVA on neonatal naive CD4+T cells from C57BL / 6 or Gpr43 knockout neonatal mice (left). Following stimulation, changes in Thl polarization rate are shown (right) (n=3 each). The schematic figure was generated using BioRender. (B) Schematic depicting the design of in-vitro experiments to examine the effects of TVA on neonatal naive CD4+T cells with or without 8-Bmoro-cAMP, PKA inhibitor, CREB inhibitor (left). Following stimulation, changes in Thl polarization rate are shown (right) (n=4 each). The schematic figure was generated using BioRender. (C) Top 5 transcription factor motifs identified in each subcluster of naive CD4+T cells from splenic cells of 2-week-old pups following TVA-milk feeding. (D) The motif score of CREB1 in different naive CD4+T cell subsets in 2-week-old pups’ splenic cells. (E) Relative Clef and Crebl mRNA levels in mouse neonatal naive CD4+T cells after treatment with siC / c / 'or siCrebl, respectively (n=5 each). (F) Representative chemiluminescent EMSA showing CTCF activity changes in neonatal CD4+T cells following TVA treatment, with or without 8-Br- cAMP or PKA inhibitor. (G) Promoter sums of Ctcf in each of subclusters of naive CD4+T cells in Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0035] 2-week-old pups’ splenic cells. Data are mean ± s.d. (A, B, E). Statistical analysis was performed using two-way ANOVA (A, B) or Student’s two-sided unpaired t-test (E).

[0036] Figure 16A-F. Dietary TVA for breeders has no significant effect on known immunomolecules or microbiomes in the milk. (A-D) Milk IgA (A), lactoferrin (B), IL-7 (C), and IL- 1 (D) levels of TVA or CON diet-fed breeders (n=5 each). (E) Milk microbial composition (order) of TVA or CON diet-fed breeders (n=3 each). (F) Milk microbial taxonomy abundance (species) of TVA or CON diet-fed breeders (n=3 each). Data are mean ± s.d. (A-D). Statistical analysis was performed using Student’s two-sided unpaired t-test (A-D).

[0037] Figure 17A-D. TVA-milk ameliorates immune response to influenza A viral infection in neonatal mice. (A) Body weight of pups in TVA and CON group, prior to influenza A virus challenge (10-day-old) (n=17 CON, n=14 TVA). (B) Body weight changes of pups in TVA and CON group during viral challenge (2 weeks). (C) Effects of TVA in breast milk on the clinical score of mice with lung infection by influenza A virus at 10-day-old (n=17 CON, n=14 TVA). (D) Representative Keratin 5 and pro-surfactant protein C IHC staining images of lungs from TVA or CON group 10-day-old mouse pups at 7 days post- viral infection. Data are mean ± s.d. (A) or mean ± s.e.m. (C). Statistical analysis was performed using Student’s two-sided unpaired t-test (A) or two-way ANOVA (C).

[0038] Figure 18A-L. TVA-milk promotes immune responses to lung viral infection. (A-J), Effects of TVA-milk on lung-infiltrating neonatal CD4+T cell population (A), %CD44+of CD4+(B), %CDlla+CD49d+of CD4+(C), CD8+T cell population (D), B cell population (E), NK cell population (F), macrophage population (G), DC population (H), neutrophil population (I), and monocyte population (I) (D4: n=8 CON, n=7 TVA; D7: n=7 CON, n=7 TVA; DIO: n=6 CON, n=7 TVA; n=4 no virus). (K-L) Effects of TVA-milk on lung (K) (n=5 each) and serum (L) (n=4 each) TNF-a level of 10-day-old mouse pups at 4 / 7 / 10 days post-viral infection. Data are mean ± s.d. (A-J) or mean ± s.e.m. (K-L). Statistical analysis was performed using two-way ANOVA (A- L).

[0039] Figure 19A-O. Anti-viral function of TVA-milk requires a shift toward Thl-skewing neonatal immunity. (A) Schematic depicting the experimental design. Lung tissues from pups with viral infection on CON- or TVA-milk (samples from 5 mice / group pooled) were analyzed by snRNA-seq and snATAC-seq. The figure was generated using BioRender. (B) Relative Gpr43 level among cell clusters. Red bars represent TVA increased clusters, blue bars represent TVA Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 decreased clusters, and black bars represent unchanged clusters. (C) Expression level of Gpr43 between TVA increased cell clusters and decreased cell clusters (n=10 decreased clusters, n=7 increased clusters). (D) UMAP plot displaying different subsets from snATAC-seq of lung tissue cells from pups with influenza A viral infection. (E) Cell proportion changes (TVA vs. CON) of different subsets in (D). (F) Relative level of motif Z-score of TBX21_MA0690.1 in Thl and CD8+effector T cells between TVA and CON. (G) Relative level of motif Z-score of

[0040] CREB1 MA0018.4 in Thl and CD8+effector T cells between TVA and CON. (H) Body weight of T-bet pups in TVA and CON group, prior to influenza A vims challenge (10-day-old) (n=4 CON, n=8 TVA). (I) Effects of TVA in breast milk on the clinical score of T-bet ' / _mice with lung infection by influenza A vims at 10-day-old (n=4 CON, n=8 TVA). (J) Body weight of TCRa KO pups in TVA and CON group, prior to influenza A virus challenge (10-day-old) (n=5 CON, n=8 TVA). (K) Effects of TVA in breast milk on the clinical score of TCRa KO mice with lung infection by influenza A vims at 10-day-old (n=5 CON, n=8 TVA). (L) Effects of TVA in breast milk on the survival rate of TCRa KO mice with lung infection by influenza A vims at 10-day-old (n=5 CON, n=8 TVA). (M) Body weight of Gpr43~'~ pups in TVA and CON group, prior to influenza A vims challenge (10-day-old) (n=6 CON, n=6 TVA). (N) Effects of TVA in breast milk on the clinical score of Gpr43''' mice with lung infection by influenza A virus at 10-day-old (n=6 CON, n=6 TVA). (O) Effects of TVA in breast milk on the survival rate of Gpr43~'~ mice with lung infection by influenza A vims at 10-day-old (n=6 CON, n=6 TVA). Data are mean ± s.d. (C, H, J, M) or mean ± s.e.m. (I, K, N), or Kaplan-Meier overall survival curves (1, o). Statistical analysis was performed using Student’s two-sided unpaired t-test (C, H, J, M), two-way ANOVA (I, K, N), or log -rank (Mantel-Cox) test (1, o).

[0041] Figure 20A-D. Mouse pups and human infants’ serum TVA levels correlate with cytokines involved in T cell function. (A) Analysis results of correlations between semm levels of TVA and cytokines in 2-week-old mouse pups on TVA- or control milk. Top candidates of positive (red circles) and negative (blue circles) are shown (right) (n=4 each). (B) Effects of having TVA enriched breast milk before wean (0-3 weeks old) on the body weight prior to influenza A vims challenge at 6-10 weeks old (n=9 CON to CON, n=7 TVA to CON). (C) Effects of having TVA enriched breast milk before wean (0-3 weeks old) on the clinical score with infection by influenza A vims at 6-10 weeks old (n=9 CON to CON, n=7 TVA to CON). (D) Representative chemiluminescent EMSA showing CTCF activity difference between naive CD4' T cells from Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 adult mice that were on TVA-milk or control milk during the pre- weaning period (0-3 weeks of age). Data are mean ± s.d. (B) or mean ± s.e.m. (C), Statistical analysis was performed using Student’s two-sided unpaired t-test (A, B), or two-way ANOVA (C).

[0042] Figure 21 A-E. Effects of postnatal and prenatal exposure of TVA on immune response to lung viral infection. (A) Schematic depicting the experimental design of the mouse-fostering strategy to test the feasibility (left). Results show success and feasibility with almost perfect survival rates of pups after the switch. The figure was generated using BioRender. (B) Schematic depicting the experimental design of mouse-fostering strategy to test The effects of TVA on protecting neonatal mice from influenza A virus infection through pre- or post-natal. The figure was generated using BioRender (left). Effects of TVA exposure in breast milk or during pregnancy on the survival rate of mice with lung infection by influenza A virus at 10-day-old (right). (C) Body weight of pups in each group of mouse-fostering experiment, prior to influenza A virus challenge (10-day-old). (D) Effects of TVA exposure in breast milk or during pregnancy on the clinical score of mice with lung infection by influenza A virus at 10-day-old. (E) TVA-milk but not during pregnancy enhanced anti-viral immunity of pups with reduced viral loads in lungs assessed by decreased influenza virus PR8 NS1 mRNA levels on Day 7 post-infection to pups at 10-day-old (n= 7 CON to CON, n=7 CON to TVA, n=6 TVA to CON, n=5 TVA to TVA). Data are Kaplan- Meier overall survival curves (A, B), mean ± s.d. (C, E) or mean ± s.e.m. (D). Statistical analysis was performed using log-rank (Mantel-Cox) test (B), one-way ANOVA (C, E), or two-way ANOVA (D).

[0043] Figure 22A-D. Effects of postnatal pr prenatal exposure of TVA on lung-infiltrating neonatal immune cells in response to viral infection in lungs. (A) Effects of TVA exposure in breast milk or during pregnancy on lung-infiltrating neonatal CD4+ T cell percentage, %CD69+of CD4+, %CD1 la+CD49d+of CD4+, %CD44+of CD4+, and peptide NP331-325 (viral nucleocapsid (N) protein)- specific cells. (B) Effects of TVA exposure in breast milk or during pregnancy on lunginfiltrating neonatal CD8+T cell population (left), activation (CD69, middle) and virus-recognizing activity (PA224-233-specific, right). (C) Effects of TVA exposure in breast milk or during pregnancy on lung-infiltrating neonatal NK cell population, B cell population, and macrophage population. (D) Effects of TVA exposure in breast milk or during pregnancy on lung-infiltrating neonatal DC population, neutrophil population, and monocyte population. n= 7 CON to CON, n=7 CON to TVA, n=6 TVA to CON, n=5 TVA to TVA. Data are mean ± s.d. (A-D). Statistical analysis was Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 performed using one-way ANOVA (A-D).

[0044] Figure 23. Cartoon schematic representation of the relationship between maternal dietary TVA and offspring outcomes.

[0045] DEFINITIONS

[0046] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.

[0048] As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise.

[0049] As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of’ and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of’ denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of’ and / or “consisting essentially of’ embodiments, which may alternatively be claimed or described using such language. Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0050] As used herein, the term “subject” broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, fish, crustaceans, etc.). As used herein, the term “patient” typically refers to a subject that is being treated for a disease or condition.

[0051] As used herein, the terms “trans-vaccenic acid” or “TV A” refer to a compound of the formula:

[0052] As used herein, the terms “cis-11,14-Eicosadienoic acid” or “EDA” refer to a compound of the formula:

[0053] As used herein, the terms “nervonic acid” or “NVA” refer to a compound of the formula:

[0054] As used herein, the term “alkyl” refers to a radical of a straight or branched saturated hydrocarbon chain. The alkyl chain can include, e.g., from 1 to 24 carbon atoms (C1-C24 alkyl), 1 to 16 carbon atoms (C1-C16 alkyl), 1 to 14 carbon atoms (C1-C14 alkyl), 1 to 12 carbon atoms (Ci- C12 alkyl), 1 to 10 carbon atoms (C1-C10 alkyl), 1 to 8 carbon atoms (Ci-Cs alkyl), 1 to 6 carbon atoms (Ci-Ce alkyl), 1 to 4 carbon atoms (C1-C4 alkyl), 1 to 3 carbon atoms (C1-C3 alkyl), or 1 to 2 carbon atoms (C1-C2 alkyl). Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n- nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0055] As used herein, the term “alkylene” refers to a divalent alkyl group.

[0056] As used herein, the term “alkenyl” refers to a radical of a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond and no triple bonds. The Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 double bond(s) may be located at any position(s) with the hydrocarbon chain. The alkenyl chain can include, e.g., from 2 to 24 carbon atoms (C2-C24 alkenyl), 2 to 16 carbon atoms (C2-C16 alkenyl), 2 to 14 carbon atoms (C2-C14 alkenyl), 2 to 12 carbon atoms (C2-C12 alkenyl), 2 to 10 carbon atoms (C2-C10 alkenyl), 2 to 8 carbon atoms (C2-C8 alkenyl), 2 to 6 carbon atoms (C2-C6 alkenyl), 2 to 4 carbon atoms (C2-C4 alkenyl), 2 to 3 carbon atoms (C2-C3 alkenyl), or 2 carbon atoms (C2 alkenyl). Representative examples of alkenyl include, but are not limited to, ethenyl,

[0057] 1 -propenyl, 2-propenyl, 1-butenyl, 2-butenyl, butadienyl, 2-methyl-2-propenyl, 3-butenyl, pentenyl, pentadienyl, hexenyl, heptenyl, octenyl, octatrienyl, and the like.

[0058] As used herein, the term “alkenylene” refers to a divalent alkenyl group.

[0059] As used herein, the term “alkynyl” means a radical of a straight or branched hydrocarbon chain containing at least one carbon-carbon triple bond. The alkynyl chain can include, e.g., from 2 to 24 carbon atoms (C2-C24 alkynyl), 2 to 16 carbon atoms (C2-C16 alkynyl), 2 to 14 carbon atoms (C2-C14 alkynyl), 2 to 12 carbon atoms (C2-C12 alkynyl), 2 to 10 carbon atoms (C2-C10 alkynyl), 2 to 8 carbon atoms (C2-C8 alkynyl), 2 to 6 carbon atoms (C2-C6 alkynyl), 2 to 4 carbon atoms (C2-C4 alkynyl), 2 to 3 carbon atoms (C2-C3 alkynyl), or 2 carbon atoms (C2 alkynyl). The triple bond(s) may be located at any position(s) with the hydrocarbon chain. Representative examples of alkynyl include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,

[0060] 2-butynyl, and the like.

[0061] As used herein, the term “alkynylene” refers to a divalent alkynyl group.

[0062] As used herein, the term “alkoxy” refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0063] As used herein, “aryl” refers to a radical of a monocyclic, bicyclic, or tricyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 1471 electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms (“C6-C14 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“Ce aryl”; i.e., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl”; e.g., anthracenyl and phenanthrenyl).

[0064] As used herein, the term “halogen” or “halo” refers to F, Cl, Br, or I. Attorney docket No. UCHI-43648.601

[0065] Client Ref. No. 24-T-025

[0066] As used herein, “heteroaryl” refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 it electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl / heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). Exemplary 5- mcmbcrcd heteroaryl groups containing one hctcroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6- membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0067] As used herein, the term “hydroxy” or “hydroxyl” refers to an -OH group.

[0068] When a group or moiety can be substituted, the term “substituted” indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments 1, 2, or 3; and in other embodiments 1 or 2) hydrogens on the group indicated in the expression using “substituted” can be replaced with a selection of recited indicated groups or with a suitable substituent group known to those of skill in the art (e.g., one or more of the groups recited below), provided that the designated atom’s normal valence is not exceeded. Substituent groups include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkenyl, guanidino, halo, haloalkyl, haloalkoxy, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, thiol, thione, or combinations thereof.

[0069] The term “supplement” as used herein refers to a nutritional product that provides nutrients (e.g. vitamins, minerals, fatty acids (e.g., TVA, EDA, NVA)) to a subject that may otherwise not be consumed in sufficient quantities by the subject. Supplements may be, for example, provided in the form of a pill, a tablet, a lozenge, a chewy capsule or tablet, a capsule, or a powder supplement that can be, for example, dissolved in water or a beverage (e.g., milk), or sprinkled on food. Supplements typically provide one or more selected compounds (e.g., TVA, EDA, NVA) without providing a significant portion of the overall nutritional needs of a subject.

[0070] The terms “infant formula” or “baby formula” as used herein refer to a powdered or liquid composition formulated to supplement or meet the nutritional needs of infants and babies (e.g., under 12 months of age).

[0071] The terms “follow-on formula (FOF)” as used herein refer to a powdered or liquid composition formulated to supplement or meet the nutritional needs of toddlers (e.g., ages 1 to five years of age) or developmentally or immunologically delayed pediatric subjects (e.g., under 14 years of age).

[0072] The term “pharmaceutical formulation” as used herein refers to a composition comprising at least one pharmaceutically-active agent, chemical substance or drug. The pharmaceutical formulation may be in solid or liquid form and can comprise at least one additional active agent, carrier, vehicle, excipient or auxiliary agent identifiable by the skilled person. The pharmaceutical formulation may be in the form of a tablet, capsule, granules, powder, liquid or syrup. Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0073] The term “effective dose” or “effective amount” refers to an amount of an agent, e.g., a neutralizing antibody, that results in the reduction of symptoms in a patient, treatment of prevention of a disease or condition, or results in a desired biological outcome.

[0074] As used herein, the terms “administration” and “administering” refer to the act of giving a drug, prodrug, or other agent, or therapeutic to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

[0075] As used herein, the terms “co-administration” and “co-administering” refer to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the coadministration of two or more agents or therapies is concurrent. In other embodiments, a first agcnt / thcrapy is administered prior to a second agcnt / thcrapy. Those of skill in the art understand that the formulations and / or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co- administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and / or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.

[0076] As used herein, an “immune response” refers to the action of a cell of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, neutrophils, etc.) and soluble macromolecules produced by any of these cells or the liver (e.g., antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and / or elimination from a subject of invading pathogens, cells or tissues infected with pathogens, or cancerous cells or other abnormal / diseased-associated cells. Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0077] DETAILED DESCRIPTION

[0078] Provided herein are compositions and methods for enhancing immune development and responses in newborns. In particular embodiments, pre-natal and post-natal supplements and or infant formula and or follow-on formula (FOF) are provided comprising long chain fatty acids (LCFAs) and very long chain fatty acids (VLCFAs), such as trans-vaccenic acid (TVA), cis- 11,14-Eicosadienoic acid (EDA), and nervonic acid (NVA), or derivatives thereof.

[0079] Maternal nutrition is a critical determinant of human breast milk (HBM) composition, which is pivotal for breastfeeding to help build an infant’s immune system and lower the risk of infections and dysfunctional organ development. HBM contains nutritional components such as fatty acids, carbohydrates, and vitamins, as well as immunomodulatory substances including infection-protecting antibodies, anti-inflammatory cytokines, and bacteria for establishment of healthy microbial gut colonization for effective immunological responses against infection and inflammation in newborns. Maternal intake of dietary fatty acids directly impacts the composition and levels of fatty acids in HBM, which can be critical for immune development in infants by playing a nutritional role and influencing the gut microbiome.

[0080] TVA is a naturally occurring trans-fatty acid, which is the predominant type of trans-fatty acids in HBM. TVA is enriched in HBM only through maternal diet because the human body cannot produce TVA. TVA in humans is mainly from ruminant-derived food including beef, lamb, milk, and butter, but naturally less than 20% of dietary TVA are metabolized by the human body. Circulating diet-derived TVA enhances adult CD8+effector T cell function and anti-tumor immunity. TVA can function as an immunomodulator and inactivate CD8+T cell-surface receptor GPR43, an immunomodulatory G protein-coupled receptor (GPCR), leading to activation of the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP- response element-binding protein (CREB) axis to reprogram adult CD8+T cells.

[0081] Experiments conducted during development of embodiments herein demonstrate that maternal dietary TVA can be enriched in human and mouse milk, leading to increased TVA levels in serums of infants and mouse pups, respectively. TVA through breastfeeding primes the readiness of neonatal naive T cells and exerts a pronounced effect on adaptive immune responses in multiple contexts, through a unique GPR43- CCCTC-binding factor (CTCF) axis, independent of CREB that is crucial in effector T cells. The neonatal immune system leans toward a “biased” Th2 immune responses by producing cytokines including IL-4 to promote antibody production Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 by B cells, rather than differentiation to Thl cells that are critical in cellular immune responses, including producing cytokines such as IFN-g for activation of cytotoxic T cells. Experiments conducted during development of embodiments herein demonstrated that maternal TVA through breastfeeding enacts a shift of neonatal immune responses from Th2-biased towards Thl- skewing, leading to augmented adaptive immunity against viral infection.

[0082] Experiments conducted during development of embodiments herein demonstrated maternal dietary TVA enriched in HBM as a previously unrecognized immunomodulator with direct, vital, and specific effects on neonatal CD4+ and CD8+ T cell development, leading to improved readiness of neonatal T cells in response to viral infection. The mechanistic study establishes a previously unknown link between maternal nutrition and neonatal Thl-Th2 immune response imbalance through breastfeeding, demonstrating that maternal TVA through breastfeeding enacts a neonatal immunity shift from Th2-biased toward Thl -skewing for improved neonatal adaptive immune response to viral infection (Fig. 23, upper). These findings have high clinical relevance, as they indicate that TVA levels in HMB from mothers correlate with serum TVA levels of their preterm infants, and both inversely correlate with the incidence of lung inflammation-associated BPD for the preterm infants (Fig. 23, lower).

[0083] Compared to adult T cells, neonatal T cells show slower responses to influenza vims infection with defective migration into the infected lungs. Experiments conducted during development of embodiments herein demonstrate that TVA in breast milk not only primes readiness of neonatal T cells but also promotes a shift to neonatal Thl polarization, which are crucial for early lung-infiltration to swiftly and effectively clear viruses from infected lungs, leading to a mitigated mortality rate of mouse neonates upon viral infection. In addition, adult mice exhibit a rather strong Thl response to influenza compared to neonatal mice, which may drive severe pro-inflammatory lung damage. In contrast, enhancing neonatal Thl responses not only boosts viral clearance without causing adult-like inflammation but also diminishes secondary inflammatory signals, thereby reducing overall inflammation and optimizing viral control in neonates. This data demonstrates that TVA-milk recalibrates the neonatal immune response to virus toward a more Thl dominant profile with reduced organ damage and inflammation, revealing an advantage of TVA in maternal nutrition and breastfeeding that help shield neonates from infection and the ensuing inflammation-driven organ damage. Experiments conducted during development of embodiments herein demonstrate cell type- Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 specific functional and regulatory differences between neonatal naive and effector T cells.

[0084] Experiments conducted during development of embodiments herein demonstrate that inclusion of TVA, EDA, and / or NVA in milk or formula provided to infants enhances immune development and immune responses. In some embodiments, pre-natal and / or post-natal (e.g., while breastfeeding) supplementation of TVA, EDA, NVA, and / or derivatives thereof to mothers and / or inclusion of TVA, EDA, NVA, and / or derivatives thereof in formula increases the amount of TVA, EDA, and / or NVA taken in by newborns.

[0085] In some embodiments provided herein are supplements (e.g., pre-natal, post-natal, etc.) comprising one or more of TVA, EDA, NVA, and / or derivatives thereof. In some embodiments, supplements herein comprise TVA, EDA, NVA, and / or derivatives thereof in addition to one or more of folate, calcium vitamin A, riboflavin, iodine, DHA, omega-3 fatty acids (e.g., docosahexaenoic acid (DHA)), pyridoxine, iron, choline, niacin, thiamin, magnesium, selenium, vitamin D, vitamin C, vitamin B12, vitamin E, and zinc.

[0086] In some embodiments, provided herein is infant formula and / or baby formula and / or follow-on formula (FOF) comprising one or more of TVA, EDA, and / or NVA. In some embodiments, the infant formula and / or baby formula and / or or follow-on formula (FOF) herein comprises TVA, EDA, NVA, and / or derivatives thereof in addition to one or more of whey (e.g., purified cow’s milk whey), casein (e.g., purified cow’s milk casein), whey protein isolate, skim milk powder, vegetable oil(s), animal milk fats, milk oligosaccharides, iron, docosahexaenoic acid (DHA), arachidonic acid (ARA), folate, amino acids, taurine, and / or other vitamins and minerals.

[0087] Provided herein are compositions (e.g., supplements, infant / baby formula, etc.) comprising TVA, EDA, NVA, and / or derivatives thereof. In some embodiments, TVA / EDA / NVA-containing or compositions are provided for the enhancement of immune development or immune response in an infant subject.

[0088] In some embodiments, compositions (e.g., supplements) comprising TVA, EDA, NVA, and / or derivatives thereof are formulated for administration to a subject (e.g., a pregnant or nursing female subject). In some embodiments, TVA, EDA, NVA, and / or derivatives thereof is formulated as a supplement. In some embodiments, provided herein is a supplement consisting of TVA, EDA, NVA, and / or derivatives thereof and suitable carriers. In some embodiments, the supplement comprises TVA, EDA, NVA, and / or derivatives thereof and other nutrients, Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 vitamins, and / or minerals. In some embodiments, other components of a supplement are present to assist or enable delivery of TVA, EDA, NVA, and / or derivatives thereof. In some embodiments, other components of a supplement are present to enhance the health of a subject. Nutrients that may be provided with TVA, EDA, NVA, and / or derivatives thereof include, but are not limited to minerals (e.g., iron, manganese, magnesium, copper, calcium, phosphorous, chromium, fluoride, selenium, sodium, chloride, potassium, zinc, copper, molybdenum, iodine, etc.), vitamins (e.g., biotin, choline, folate, niacin, pantothenic acid, riboflavin, thiamin, biotin, inositol, and vitamins A, Bl, B6, pyridoxine, B12, C, D, E, K, etc.), protein, protein hydrolysates and amino acids (e.g., tyrosine, glutamine, proline, leucine, leucine, isoleucine, threonine, lysine, cysteine, histidine, methionine, phenylalanine, threonine, tryptophan, valine, histidine, taurine, L-camitine, etc), other fatty acids (e.g., omega-3 fatty acids (e.g., a-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), etc.), omega-6 fatty acids (e.g., linoleic acid (LA), arachidonic acid (ARA) etc.), omega-9 fatty acids (e.g., erucic acid, etc), trans fatty acids, saturated fatty acids (e.g., lauric acid, myristic acid, etc), unsaturated fatty acids, polyunsaturated fatty acids (PUFA), mono unsaturated fatty acids (MUFA), long chain fatty acids, short chain fatty acids, etc.), and other biomolecules (carbohydrates (e.g., lactose, sucrose, glucose, fructose, maltodextrins, starches, non-digestible oligosaccharides), probiotics, synbiotics, nucleotides, nucleosides, phospholipids, triacylglycerols). In some embodiments, a supplement comprises TVA, EDA, NVA, and / or derivatives thereof and one or more active bacterial cultures, wherein the bacterial cultures comprise species of bacteria that are beneficial to human health and / or enhancing immune development and responses in newborns. In some embodiments, a TVA-containing, EDA-containing, and / or NVA-containing supplement is formulated according to standard supplement formulations that are understood in the art.

[0089] In some embodiments, a TVA-containing, EDA-containing, and / or NVA-containing composition is formulated as a pharmaceutical composition. In some embodiments, TVA, EDA, NVA, and / or derivatives thereof and any co-formulated agents (when present) are provided in formulations for administration to a subject by a suitable route. The pharmaceutical formulations described herein can be administered to a subject by multiple administration routes, including but not limited to, oral administration. Moreover, TVA-containing, EDA-containing, and / or NVA- containing pharmaceutical compositions are formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 aerosols, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, and capsules.

[0090] Pharmaceutic al / supplemental preparations comprising TVA, EDA, NVA, and / or derivatives thereof are provided for oral use can be obtained by mixing one or more solid excipients with TVA, EDA, NVA, and / or derivatives thereof (and other active agents desired in the formulation) with any suitable substituents and functional groups disclosed herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets, pills, or capsules. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents may be added, such as the cross-linked croscarmcllosc sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0091] In some embodiments, routes of administration, formation of TVA, EDA, NVA, and / or derivatives thereof are selected to provide efficient and effective delivery. In some embodiments, TVA, EDA, NVA, and / or derivatives thereof is provided with a suitable carrier. In some embodiments, TVA, EDA, NVA, and / or derivatives thereof is encapsulated of embedded into a carrier. In some embodiments, a carrier may comprise a liposome, nanoparticle, or other suitable system for delivery of TVA, EDA, NVA, and / or derivatives thereof. In some embodiments, the carrier and / or delivery system is selected to optimize the solubility, stability, bioavailability, targeting, etc. of TVA, EDA, NVA, and / or derivatives thereof.

[0092] The supplement or pharmaceutical compositions described herein may be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of TVA, EDA, NVA, and / or derivatives thereof. The unit dosage may be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Alternatively, multiple-dose reclosable containers can be used, in which case it is typical to include a preservative in the composition. Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0093] Dosing and administration regimes may be tailored by the clinician, or others skilled in supplements or the pharmacological arts, based upon well-known pharmacological and therapeutic considerations including, but not limited to, the desired level of therapeutic effect, and the practical level of therapeutic effect obtainable. For compositions that have relatively little or no dose-related toxicity considerations, and where maximum efficacy is desired, doses in excess of the average required dose are not uncommon. This approach to dosing is commonly referred to as the “maximal dose” strategy. In certain embodiments, TVA, EDA, NVA, and / or derivatives thereof is administered to a subject at a dose of about 0.01 mg / kg to about 200 mg / kg (e.g., 0.01 mg / kg, 0.02 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 50 mg / kg, 100 mg / kg, 200 mg / kg, or ranges therebetween). Dosing may be once per day, multiple times per day (e.g., 2, 3, 4, etc.), once per week, or according to any suitable protocol. In some embodiments, TVA, EDA, NVA, and / or derivatives thereof is administered a single time, each time a co-administered agent is delivered, or for a time period of days, weeks, months, indefinitely, etc.

[0094] In some embodiments, compositions comprising TVA, EDA, NVA, and / or derivatives thereof are administered alone or in combination with any other nutrients, minerals, food source, etc. using standard delivery systems and methods, and in at least some aspects, together with a pharmaceutically acceptable carrier or excipient.

[0095] In some embodiments, compositions (e.g., infant / baby formula) comprising TVA, EDA, NVA, and / or derivatives thereof are formulated for administration to a subject (e.g., a newborn or infant or pediatric). In some embodiments, TVA, EDA, NVA, and / or derivatives thereof is formulated as infant / baby formula. In some embodiments, provided herein is infant / baby formula consisting of TVA, EDA, NVA, and / or derivatives thereof and suitable carriers. In some embodiments, the supplement comprises TVA, EDA, NVA, and / or derivatives thereof and other nutrients, vitamins, and / or minerals. In some embodiments, other components of a supplement are present to assist or enable delivery of TVA, EDA, NVA, and / or derivatives thereof. In some embodiments, other components of a supplement are present to enhance the health of a subject. Nutrients that may be provided with TVA, EDA, NVA, and / or derivatives thereof include, but are not limited to minerals (e.g., iron, manganese, magnesium, copper, calcium, phosphorous, etc.), vitamins (e.g., biotin, choline, folate, niacin, pantothenic acid, riboflavin, thiamin, and vitamins A, B6, B12, C, D, E, K, etc.), and other fatty acids (e.g., omega-3 fatty acids (e.g., a- Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), etc.), omega- 6 fatty acids (e.g., linoleic acid (LA), etc.), trans fatty acids, saturated fatty acids, unsaturated fatty acids, polyunsaturated fatty acids (PUFA), etc.).

[0096] In some embodiments, exemplary derivatives of TVA, EDA, and NVA that may find use in any embodiments herein are depicted in figures 6, 7, and 8. In some embodiments, suitable derivatives may differ in length from TVA, EDA, or NVA by one or more carbons (e.g., 1, 2, 3, 4, 5, 6, etc.). In some embodiments, suitable derivatives may differ in the number (e.g., 0, 1, 2, 3, etc.) or location of unsaturated carbons (i.e., double bonds) from TVA, EDA, or NVA. In some embodiments, suitable TVA, EDA, or NVA derivatives may contain additional substituents (e.g., methyl, ethyl, propyl, -OH, — NH2, phenyl, etc.).

[0097] Exemplary derivatives of TVA include isomers with the double bond shifted, such as C18:l trans-10 or C18:l trans-12, as well as C18:2 trans-11, cis-13, a conjugated diene form. Chain length variants of TVA include C17:l trans-9 (shortened by one carbon) or C20:l trans- 13 (elongated by two carbons), which preserve the core trans-monounsaturated structure. Suitable TVA derivatives may comprise substituents, such as a methyl group at position 11, forming 11- methyl TVA, or a hydroxyl group at position 12 to yield 12-hydroxy TVA. Other functional substitutions include 11-amino TVA and 11-phenyl TVA. Other similar TVA derivatives will be understood.

[0098] Exemplary derivatives of EDA include positional isomers such as C20:2 cis-12,15, and C20:2 cis-9,12 (the latter being identical to linoleic acid). Additional double bonds can be added to generate C20:3 cis-8, 11,14, a triene. Chain-modified analogs include C18:2 cis-9,12 (shortened) and C22:2 cis-13, 16 (elongated). Structural diversity can also be achieved via small substituents like a methyl group at position 13, forming 13-methyl EDA, or a hydroxyl group at position 14, forming 14-hydroxy EDA. Additional variants include ethyl substitution at CIO and phenyl substitution at C13, which introduce bulk or aromaticity near the unsaturated region. Other similar EDA derivatives will be understood.

[0099] Exemplary derivatives of NVA include isomers with the double bond shifted slightly, such as C24;l cis-13 or C24;l cis-17, and polyunsaturated analogs like C24;2 cis- 15, 18. Chainlength valiants include C22:l cis-13, a shortened co-9 analog, and C26:l cis-17, which retains the core features while increasing chain length. Small substituents provide further diversity, including a methyl group at C15 (15-methyl NVA), a hydroxyl group at C16 (16-hydroxy NVA), Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 or an amino group at the double bond site (15-amino NV A). Additional modifications such as a phenyl group at C15 or a propyl group at C12 can add steric or functional complexity while retaining the very-long-chain monounsaturated backbone. Other similar NVA derivatives will be understood.

[0100] EXPERIMENTAL

[0101] Results

[0102] Maternal TVA reshapes neonatal T cell development via breastfeeding

[0103] Experiments were conducted during development of embodiments herein to investigate whether maternal dietary TVA, which can be enriched in HBM as the predominant naturally occurring trans-fatty acid, contributes to infant immune system development and neonatal immune responses. It was found that TVA levels in HBM samples from mothers correlate to the TVA levels in scrum samples on the blood spot cards from their preterm infants (Fig. 9A). A mouse model was developed to test effects of maternal dietary TVA on neonatal immune system development. It was found that lactating mouse dams in two groups on diets supplemented with 0.1% or 1% TVA since pregnancy (Fig. 9B) produced milk with accumulated TVA to comparable levels to each other, but TVA was not detected in milk from a group of dams on a normal control diet (Fig. 9C). In addition, TVA accumulated in the serum of pups on TVA- enriched milk in a time-dependent manner until weaning (3 weeks post-birth; on TVA-enriched diet afterwards), whereas no serum TVA was detected in pups on control milk (Fig. 9D). However, pups on TVA-milk or control milk had comparable body weights (Fig. 13A), indicating that TVA in milk likely has a minimal nutritional role in neonatal mouse body. Increased spleen size was found in pups on TVA-milk compared to pups on control milk at the age of 2-3 weeks, but the spleen size became comparable at the age of 5 weeks (Fig. 9E). The pups on TVA-milk at the age of 2 weeks had significantly elevated cell numbers of total splenocytes, CD4+ and CD8+ T cells, B cells, neutrophils, dendritic cells (DCs), and monocytes in spleens (Fig. 13B) with only significantly increased frequency of CD4+ T cells (Fig. 13C), compared to control pups. In contrast, cell numbers of total splenocytes and most immune cell types including T cells were comparable between pups on TVA-milk and control pups at the age of 5 weeks (Fig. 13D), with significantly increased CD8+ T cell frequency but reduced Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 percentages of B cells and DCs (Fig. 13E). Further analysis revealed significantly increased cell numbers of both CD4+ and CD8+ T cells (Fig. 13F-G) with elevated frequency of naive CD4+ T cells (Fig. 13H) in spleens of pups on TVA-milk compared to control pups at the age of 2 weeks, whereas other tested T cell subsets were comparable (Fig. 13H-K) except for increased expression levels of T cell activation marker CD69 on CD8+ T cells (Fig. 13K). Pups on TVA- milk at the age of 2 weeks also had increased thymus weights (Fig. 13L), with significantly reduced frequency of CD4 / CD8 double-negative (DN) precursors but increased frequency of later stage CD4 / CD8 double-positive (DP) thymocytes, whereas thymotic percentages of both CD4+ (4SP) and CD8+ (8SP) cells were comparable between pups on TVA-milk and control pups (Fig. 13M). These results indicate that TVA-milk promotes expansion of neonatal naive CD4+ T cells in spleens and maturation of thymocytes, leading to accelerated generation of functional T cells in neonatal mouse pups.

[0104] To comprehensively analyze the effects of breastfeeding TVA on neonatal immune cells, single-nuclei multi-omics wc performed, including single-nuclei RNA sequencing (sn-RNA-scq) and single-nuclei Assay for Transposase-Accessible Chromatin using sequencing (sn-ATAC- seq) to characterize transcriptional profiles and chromatin accessibility of splenic cells of pups on either TVA-milk or control milk at the age of 2 weeks (Fig. 9F). Analysis of the transcriptional and chromatin landscapes of splenic cells at the single-cell level identified 20 distinct clustered populations that include different subsets of T cells, B cells, and myeloid cells (Fig. 9F). It was found that pups on TVA-milk had increased splenic populations of all identified T cell subsets including naive CD4+ T cells (C3) that represents the most expanded population, along with naive CD8+ T cells (C4), CD8+ T cells (C6 and CIO), CD4+ effector T cells (C9), CD8+ effector T cells (C15), and y5 T cells (C20), as well as mature follicular B cells (C5) and dendritic cells (Cl 8), all of which are crucial for T cell-dependent adaptive immunity, compared to the control pups (Fig. 1G).

[0105] In addition, increased promoter sums of representative genes related to T cell maturation and function were identified, including I12rb, Tnfrsf9, Ifng, Gzmb, and Icos, proliferation and survival including Bcl2, Bell lb, and Cd28, and migration and trafficking including Ccl5, Cxcr6, and Ccr8 (Fig. 9H) in the splenic T cells of pups on TVA-milk compared to the control pups. Notably the promoter sums of Gpr43 (Ffar2), which was identified as a TVA target in adult CD8+ effector T cells, are increased in clusters of T cells that are expanded in pups on TVA- Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 milk compared to control pups, including naive CD4+ T cells (C3), naive CD8+ T cells (C4), CD4+ effector T cells (C9), CD8+ T cells (CIO), and CD8+ effector T cells (C15) (Fig. 91). In addition, transcription factor motifs of CREB and TBX21 (T-bet) are increased in TVA- expanded neonatal naive CD4+ T cells (C3) (Fig. 14A-B). These data together indicate that TVA-milk primarily promotes neonatal T cell development, especially on the naive CD4+ T cell compartment.

[0106] TVA-milk reprograms neonatal CD4+ naive T cells through a GPR43-CTCF axis

[0107] To determine whether TVA in breast milk functions in neonatal T cells through cellsurface receptor GPR43(16), experiments were conducted during development of embodiments herein to examine the effects of Gpr43 -deficiency on neonatal naive CD4+ T cells (Fig. 10A, left), which represent the most expanded population in spleens of pups on TVA-milk (Fig. 9G) and play a crucial role in adaptive immunity. TVA-milk significantly increased ex-vivo abilities of neonatal CD4+ T cells in terms of Thl polarization, activation assessed by CD69 expression, and cell expansion (Fig. 10A, right) but had minimal effects on Th2 and Thl7 polarization (Fig. 14C). In contrast, cells from Gpr43- / - pups on control milk showed elevated abilities of Thl polarization, activation, and cell expansion compared to cells from control pups, suggesting an immunosuppressive role of GPR43 in neonatal CD4+ T cells, whereas TVA-milk had minimal effects on neonatal CD4+ T cells in Gpr43- / - pups (Fig. 10A, right).

[0108] Experiments conducted during development of embodiments herein demonstrated that in vitro TVA treatment enhanced Thl polarization of neonatal naive CD4+ T cells but had no effects on cells from Gpr43- / - pups (Fig. 15A). Consistent with findings that TVA inactivates GPR43 to increase cAMP production and PKA activity, treatment with cell permeable 8-Bmoro- cAMP and PKA inhibitor resulted in increased and decreased Thl polarization of neonatal naive CD4+ T cells, respectively, and both abolished the effects of TVA (Fig. 15B). However, treatment with CREB inhibitor had minimal effects on Thl polarization of neonatal naive CD4+ T cells, nor did it affect TVA-enhanced Thl polarization (Fig. 15B). These data indicate that TVA reprograms neonatal naive CD4+ T cells through the GPR43-cAMP-PKA axis, which, however, is CREB-independent.

[0109] To identify the transcription factor that is responsible for the effects of TVA on neonatal naive CD4+ T cells, experiments were conducted during development of embodiments herein to Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 investigate 4 subclusters with distinct patterns of transcription factor activity in splenic neonatal naive CD4+ T cell population (C3 in Fig. 9F) in pups on TVA-milk compared to control pups (Fig. 10B-C). Among the top 5 transcription factor motifs identified in each subcluster, CTCF in subcluster 1 represents the only transcription factor with significantly increased activity (Fig. 2D, Fig. 15C), whereas minimal changes of CREB were detected in all 4 subclusters (Fig. 15D). CTCF (CCCTC-binding factor) is a zinc finger transcription factor that plays a crucial role in maintaining the expression of key genes and chromatin architecture by regulating the three- dimensional genome organization and preventing improper interactions between enhancers and promoters. CTCF cooperates with the lineage-specific transcriptional factor TBX21 (T-bet) that is critical for Thl polarization to ensure proper gene expression in Thl cells including Ifng. Knockdown of Ctcf, but not Crebl (Fig. 15E), abolished Thl polarization and TBX21 (T-bet) activation enhanced by either TVA or 8-Bmoro-cAMP in neonatal naive CD4+ T cells (Fig.

[0110] 10E). Moreover, treatment with TVA significantly enhanced CTCF transcription factor activity assessed by increased DNA binding ability in neonatal naive CD4+ T cells, which was not further enhanced by 8-Bmoro-cAMP and could be abolished by treatment with a PKA inhibitor (Fig. 10F, Fig. 15F). Since the promoter sums of Ctcf are not altered in subclusters of naive CD4+ T cells (Fig. 15G), these results indicate that TVA reprograms neonatal naive CD4+ T cells through a unique GPR43-cAMP-PKA-CTCF axis, likely by enhancing CTCF transcription factor activity but not its expression.

[0111] It was also confirmed that supplementation of dietary TVA to mouse dams did not alter the major components in their milk. Specifically, the levels of representative IgA, lactoferrin, and cytokines including IL- 10 and IL-7 in milk from dams on TVA-diet were comparable to those in milk from control dams (Fig. 15A-D). In addition, the diversity and patterns of microbial distribution in mouse milk varied widely without a clear trend among samples from different mice in the same group of dams, indicating that feeding TVA-enriched diet to mouse dams did not alter the milk microbiome with a noticeable trend (Fig. 15E-F).

[0112] TVA-milk primes the readiness of neonatal T cells against viral infection

[0113] To determine the functional effect of maternal TVA supplementation on neonatal immunity, a neonatal mouse model of lung infection was used with the H1N1 influenza A virus A / Puerto Rico / 8 / 1934 via intranasal administration (Fig. 11 A). Although pups at the age of 10 Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 days showed comparable body weights (Fig. S5A), pups on TVA-milk had significantly enhanced survival (Fig. 3B) and reduced clinical score (Fig. S5B-C) in response to influenza A virus. Moreover, pathologic analysis of lung tissues from control pups showed that infection by influenza A virus resulted in increased lung epithelial damage assessed by immunohistochemistry (IHC) staining of Keratin 5(24) (Fig. S5D, upper, quantitative results shown in Fig. 3C, left) with reduced normal epithelium assessed by IHC staining of prosurfactant protein C(24) (Fig. S5D, lower, quantitative results shown in Fig. 3C, right), compared to control pups without viral treatment. In contrast, pups on TVA-milk showed significantly reduced lung damage and increased normal epithelium, compared to control pups with viral infection (Fig. 17D, quantitative results shown in Fig. 11C).

[0114] Further analysis of lung tissues revealed that pups on TVA-milk had significantly reduced viral loads in lungs assessed by decreased influenza virus PR8 NS1 mRNA levels on as early as Day 7 and Day 10 post-infection, compared to control pups (Fig. 1 ID). Moreover, although the frequency of lung-infiltrating CD4+ T cells were not significantly altered in pups on TVA-milk (Fig. 18 A), these cells had a significantly increased activation state assessed by elevated CD69 and CD44 levels (Fig. HE, left, Fig. 18B) on as early as Day 7 post-infection, compared to control pups. In addition, the frequency of influenza- specific CD4+ T cells accessed as CDl la+ / CD49d+- (Fig. 18C) and NP331-325+ (epitope of viral N protein)-specific cells (Fig. 1 IE, right) were also significantly elevated on Day 7 but reduced on Day 10 post-infection, whereas these populations peaked on Day 10 post-infection in control pups. Similar results were obtained for lung-infiltrating CD8+ T cells with significantly increased frequency (Fig. 18D) and CD69 expression (Fig. 1 IF, left) in TVA-breastfeeding pups on as early as Day 7 post-infection, compared to control pups. The frequency of influenza-specific (PA224-233+) CD8+ T cells was significantly elevated on Day 7 but reduced on Day 10 post-infection, compared to this population in control pups that was increased on Day 10 post-infection (Fig. 1 IF, right). These data indicate that TVA in breast milk primes the readiness of neonatal T cells in immune responses to viral infection with early lung-infiltration to swiftly and effectively clear viruses from infected lungs, leading to a mitigated mortality rate of mouse neonates.

[0115] In contrast, most of the populations of lung-infiltrating B cells, NK cells, macrophages, DCs, neutrophils, and monocytes (Fig. 18E-I) were not significantly altered in pups on TVA- milk compared to control pups, while only populations of lung-infiltrating B cells and NK cells Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 were significantly increased or reduced on Day 10 post-infection, respectively, in pups on TVA- milk. Furthermore, pups on TVA-milk showed significantly reduced inflammation assessed by decreased IL-ip in lungs (Fig. 3G) and TNF-a levels in lungs and serum (Fig. 18K-L). These results together suggest that TVA-milk enhances the readiness of neonatal T cells, leading to an early but more effective response to viral infection and inflammation in lungs to improve mortality of virus-infected pups.

[0116] TVA-milk enacts a shift to Thl-skewing immunity against viral infection

[0117] Single-nuclei multi-omics analysis was performed on lung tissue cells of pups that were sacrificed on Day 7 post-viral infection (Fig. 19A). Results of snRNA-seq identified 20 distinct clustered populations (Fig. 11H). It was found that, upon viral infection, pups on TVA-milk had increased lung-infiltrating populations of diverse T cell subsets including CD8+ effector T cells (Cl, C2, C12), Thl-like T cells (C3), CD8+ cytotoxic T cells (C7), and tissue-resident-like T cells (Cl 6), but reduced lung-infiltrating Th2-likc T cells (C6) and B-lincagc cells (C8), compared to control pups (Fig. 1 II). These results indicated that maternal TVA through breastfeeding implements a shift from Th2-biased neonatal immunity to Thl-skewing, which enhances recruitment of lung infiltrating Thl cell subset to help clear viral loads more effectively in infected lungs.

[0118] Lung-infiltrating cell clusters with increased populations had higher Gpr43 mRNA levels (Fig. 19B-C). In addition, snATAC-seq analysis revealed 15 clusters (Fig. 19D), among which 4 clusters with increased cell populations in lung tissues from pups on TVA-milk with viral infection were identified as Thl cells (Cl) and CD8+ effector T cells (C4, C7, and C9 (Fig. 19E). Increased transcription factor activity of TBX21 (T-bet) with higher motif-Z scores was observed in lung-infiltrating Thl cells of pups on TVA-milk compared to control pups, whereas TBX21 activity was comparable in lung-infiltrating CD8+ effector T cell populations between pups on TVA-milk and control milk (Fig. 19F). In contrast, the transcription factor activity of CREB was increased in lung-infiltrating CD8+ effector T cell populations, but not Thl cells, of pups on TVA-milk compared to control pups (Fig. 19G). These data are consistent with the finding that TVA-milk functions through GPR43 -dependent activation of CTCF to enhance its cooperation with TBX21 in neonatal naive T cells for proper Thl gene expression.

[0119] Tbx21 knockout (Tbx21- / -) mice that cannot effectively develop Thl cells were used to Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 examine the crucial roles of TBX21 and Th 1- skewing shift in effects of maternal TVA on neonatal immunity (Fig. 3J, left). Tbx21 gene deficiency abolished anti-viral effects of TVA- milk, leading to comparable survival and clinical score between pups on TVA-milk and control milk (Fig. 11J, Fig. 19H-I). Similar results were obtained using TCR-a knockout (KO) mice deficient of aP T cells (Fig. 19J-L) and Gpr43 knockout (Gpr43- / -) mice (Fig. S7M-0). These results demonstrate a crucial role of the neonatal immunity shift from Th2-biased toward Thl- skewing enacted by TVA-milk in immune responses against viral infection and also establish a previously unknown link between maternal nutrition and neonatal Thl-Th2 imbalance in immune responses through breastfeeding.

[0120] TVA in HBM inversely correlates with incidence of preterm bronchopulmonary dysplasia

[0121] Experiments were conducted during development of embodiments herein to examine the relationship between TVA level and cytokine profile in blood of mouse pups and human infants. Scrum TVA levels of pups on TVA-milk at the age of 2 weeks positively correlated with IL-2, IL-22, and CXCL9 levels, all of which are related to T cell functions, but inversely correlated with IL-4 (Fig. 20A). Similar results were obtained using blood spot samples from preterm infants (Fig. 12A, left). The infant blood TVA levels positively and negatively correlated with blood IL-2 and IL4 levels, respectively (Fig. 12A, middle and right). Given that IL-4 is vital to induce differentiation of naive helper T cells to Th2 cells and blocking IL-4 promotes a shift from Th2-biased to Thl-skewing neonatal immune responses, this finding is consistent with results indicating that TVA-milk preferentially promotes a shift toward Thl neonatal immunity. Preterm infants have enfeebled Thl immune responses and thus are vulnerable to infections, which may cause chronic inflammation, leading to disrupted organ development with an increased risk of diseases such as bronchopulmonary dysplasia (BPD), which is strongly associated with lung inflammation. To comprehensively examine the link between human milk fatty acids and preterm- associated inflammatory BPD, a mass spectrometry-based fatty acid profiling approach was used (Fig. 12B, left). TVA levels in HBM samples from mothers as the first in rank inversely correlated with the incidence of BPD for their preterm infants, (Fig. 12B, right). Consistent with this finding, TVA levels in the blood spot samples from a group of preterm infants also inversely correlated with their incidence of BPD (Fig. 12C). These results indicate a mechanistic link between TVA in breast milk and neonatal T cell functions, which Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 likely contributes to preventing preterm inflammation-associated diseases.

[0122] Early-life exposure to TVA via breastfeeding impacts immune imprinting

[0123] Early-life immune imprinting describes how exposures to environmental cues such as microbial interactions or dietary factors during infancy and early childhood can significantly impact immune development and have long-lasting imprinting effects on immune responsiveness and disease susceptibility. Neonatal CD4+naive T cells serve as the foundation of adaptive immunity and thus play a crucial role in early-life immune imprinting and contribute to shaping of lifelong immunity. Therefore, it was examined whether postnatal exposure to TVA through breastfeeding has an immune imprinting effect. Normal control diet was fed to both groups of pups on TVA-milk and control milk after weaning at age of three weeks (TVA-CON and CON- CON, respectively) and the mouse model of lung infection was conducted with influenza A virus treatment when the mice reached adulthood at age of 6-10 weeks (Fig. 12D, upper). Pups on TVA-milk at age of 3 weeks had accumulated TVA levels in scrum, which became undetectable at adulthood after being fed on control diet post-weaning (Fig. 12E). However, these mice in the group of TVA-CON showed significantly enhanced survival (Fig. 12F) with comparable starting body weight but reduced clinical score during the infection study (Fig. 20B-C) in response to influenza viral exposure, compared to the control CON-to-CON pups. In addition, the adult naive CD4+ T cells from mice in both groups were examined (Fig. 12D, lower), and increased CTCF activity (Fig. 12G, Fig. 20D) and enhanced Thl polarization (Fig. 12H) was observed in cells from mice in the TVA-CON group, compared to mice in the CON-CON group. These results indicate that reprogramming of neonatal CD4+ naive T cells by exposure to maternal TVA through breastfeeding has a long-lasting effect on immune imprinting.

[0124] Experiments were conducted during development of embodiments herein to examine whether prenatal or postnatal TVA exposure via breastfeeding plays a predominant role in TVA- primed neonatal T cell immunity. A mouse-fostering strategy was used to assign the pups born by C57BL / 6 (black color) breeders on either TVA-enriched or control diet to FVB / NJ foster mothers (white color) on either TVA-enriched or control diet (Fig. 21 A, left). At the age of 2 weeks, only one C57BL / 6 pup fed by an FVB / NJ foster mother died in the CON-to-CON group (Fig. 21A, right), demonstrating the overall success of the mouse-fostering strategy. The neonatal mouse model of lung infection with influenza A virus treatment was performed using these pups Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0125] (Fig. 21B, left). Compared to the control CON-to-CON pups, pups in the groups of TVA-to- TVA (prenatal- and postnatal-TVA) and CON-to-TVA (postnatal-TVA) showed significantly enhanced survival (Fig. 2 IB, right) with comparable starting body weight but reduced clinical score during the infection study (Fig. 21C-D) in response to influenza viral exposure. In contrast, the group of TVA-to-CON (prenatal-TVA) had comparable survival and clinical score, compared to the control CON-to-CON pups upon viral infection (Fig. 21B-D). Consistent with these findings, the groups of TVA-to-TVA and CON-to-TVA, but not the TVA-to-CON pups, showed significantly reduced viral load in lungs (Fig. 2 IE) and increased population and activation states of lung-infiltrating CD4+ and CD8+ T cells (Fig. 22A-B), compared to the control CON-to-CON pups upon viral infections, whereas lung-infiltrating percentages of NK cells, B cells, macrophages, DCs, neutrophils, and monocytes were comparable among the four groups of pups (Fig. 22C-D). These results demonstrate that relative to prenatal TVA exposure, postnatal exposure of TVA via breastfeeding is predominantly essential to prime the readiness of neonatal T cells.

[0126] Materials and Methods

[0127] Primary cells

[0128] Mouse neonatal or adult naive T cells were isolated from the spleen and peripheral lymph nodes (PLN) of 10-14 days old or 8-12 weeks old C57BL / 6 mice by magnetic bead purification using Easy Sep™ Mouse Naive T Cell Isolation Kit according to the manufacturer’s instructions (Stem Cell Technologies). Cells were cultured in Click’s media at 37°C and 5% CO2 incubator for further experiments.

[0129] Mice

[0130] Animal experiments were conducted and designed according to protocols approved by the Institutional Animal Care and Use Committee of The University of Chicago. Mice were housed and bred at the University of Chicago Animal Resource Center in specific pathogen-free conditions. Mice were on 12-hour light / dark cycles that coincided with daylight in Chicago, IL, USA, housing facility was maintained at 20-25°C and 30-70% humidity. C57BL / 6J (The Jackson Laboratory, JAX:000664; RRID: IMSR_JAX:000664), TCRo. Knock-out (B6.129S2- TcratmlMom / J) (The Jackson Laboratory, JAX:002116; RRID: IMSR_JAX:002116), FVB / NJ Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0131] (The Jackson Laboratory, JAX: 001800; RRID: IMSR_JAX:001800), Tbx21''- (B6.129S6- Tbx21tmlGlm / J) (The Jackson Laboratory, JAX: 004648; RRID: IMSR_JAX:004648) mice were purchased from The Jackson Laboratory. Gpr43' / ' mice were kindly provided by Dr. Brian Layden. Sex-and age-matched mice were used throughout the study at 7-12 weeks old, and both male and female mice were used. The genetically modified mice were viable and developed normally.

[0132] Human samples

[0133] Microbiome In Neonatal Development (MIND) study recruits preterm infants (infants bom < 37 weeks postmenstrual age) from the University of Chicago Comer Children’s Hospital after retrieving written informed consent from a parent. Exclusion criteria included presence of genetic syndromes, severe congenital anomalies, or nonviability. All sample collection and study procedures were approved by the University of Chicago Institutional Review Board (IRB16- 1431) which abides the Declaration of Helsinki.

[0134] TVA measurement of infant blood spot card samples

[0135] The blood spot cards of the preterm infants were used for TVA level detection. Ten 3.2- mm (1 / 8-inch) diameter disks were punched from dried infant blood-spots. The disks were eluted together in 180 pL of PBS containing a "Complete protease inhibitor cocktail without EDTA" (Pierce), including 0.5 g / L sodium azide, by agitating at room temperature for 2 hours and centrifuged at 1000g for 10 minutes. The supernatant was collected for TVA extraction: 150 ml of samples was added with 3 mL of a mixture chloroform / methanol (2:1, v / v), then samples were sonicated at 4°C (20%, pulse 4s, stop 4s for Imin) and centrifuged at 4000g at 4°C for 5 min. Cell pellets were kept for a re-extraction step, and supernatants were transferred to a new test tube with 0.6 mL of H2O and shaken vigorously following centrifugation at 4000g at 4°C for 10 min. The lower layer of chloroform with extracted lipids was collected. The remaining cell pellets were re-extracted, repeating the procedure. Two supernatant fractions were combined and were evaporated under a stream of N2. Extracted lipid fraction was resuspended using 50 pl 90% Acetonitrile in H2O and 50 ml Acetone. TVA standard was diluted in 50 ml Acetone and 50 pl 90% Acetonitrile in H2O to yield 1 mM solution. For 100 pl solution extracted from CHL / Methanol, add 3 pl pyridine, 0.5 pl 0.5M EDCI, and 5 ml IM13C6-Aniline. For 100 pl Attorney docket No. UCHI-43648.601

[0136] Client Ref. No. 24-T-025

[0137] TVA solution extracted from Acetone, add 3 pl pyridine, 0.5 pl 0.5M EDC, and 1.35 ml 3.7M Aniline. The reaction was incubated at 37°C for 2 hours and mixed 20ul serum extraction from CHL / Methanol with 20 pl TVA standard derivatized solution, the mixture was then diluted in 60 pl Methanol and analyzed by LC-MS.

[0138] The mouse model to test the effects of TVA on the neonatal immune system

[0139] Establishing TVA-feeding mouse lines: For each mouse line, 8-week-old C57BL / 6J males and females are fed with the TVA-enriched diet or control diet for two weeks before pairing them for breeding. Their offspring were kept on TVA-enriched diet or control diet and inbred to continue this line. After the control diet and TVA diet mouse lines stabilized (2-3 generations), the breeders and neonatal pups of each group were used for further analysis. For mouse milk collection, breeders were separated from their 1- or 2- week-old pups for 1 hour. Following this, each breeder received a 1 IU intraperitoneal (IP) injection of oxytocin. Fifteen minutes later, milk samples were collected from the breeders. Blood samples were also collected from pups aged 1 to 5 weeks from each group. Additionally, body weight, spleen weight, and thymus weight were monitored. Spleen and thymus tissues were further analyzed for immune cell profiling. Spleen samples from 2-week-old pups in each group were also used for singlenuclei multi-omics analysis, including snRNA-seq and snATAC-seq (samples from 5 mice / group pooled).

[0140] Diet formula

[0141] Diets for mouse experiments were obtained from Research Diet Inc. Control diet (D12450J), 0.1% TVA diet (D23031402), 1% TVA diet (D19110701).

[0142] Mouse splenocytes isolation

[0143] Mouse spleens were disrupted with syringe plunger in 1 mL PBS in a 40 pm strainer and cells were filtered to a 15 mL tube, then the cells were washed with PBS, and centrifuged at 300xg for 5 minutes. The resulting cell pellets were re-suspended with 2 mL red cell lysis buffer (Invitrogen), then cells were incubated at room temperature for 10 minutes and centrifuged at 300xg for 5 minutes after adding 13 mL PBS. The resulting cell pellets were designated splenocytes and used for the following experiments. Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0144] Mouse peripheral lymph node cell isolation

[0145] Peripheral lymph nodes were disrupted with syringe plunger in 1 mL PBS in a 40 pm strainer and cells were filtered to a 15 mL tube, then the cells were washed with PBS, and centrifuged at 300xg for 5 minutes. The resulting cell pellets were designated lymph node cells and used for the following experiments.

[0146] Mouse thymus single-cell isolation

[0147] Mouse thymuses were disrupted with syringe plunger in 1 mL PBS in a 40 pm strainer and cells were filtered to a 15 mL tube, then the cells were washed with PBS, and centrifuged at 300xg for 5 minutes. The resulting cell pellets were re-suspended with 2 mL red cell lysis buffer (Invitrogen), incubated at room temperature for 10 minutes, and centrifuged at 300xg for 5 minutes after adding 13 mL PBS. The resulting cell pellets were designated thymus cells and used for the following experiments.

[0148] Primary naive CD8+or naive CD4+T cell isolation and activation

[0149] Mouse primary naive CD8+or CD4+T cells were isolated from the spleen and peripheral lymph nodes (PLN) of C57BL / 6 mice by magnetic bead purification using EasySep™ Mouse naive CD8+or CD4+T Cell Isolation Kit according to the manufacturer’ s instructions (Stemcell Technologies). Isolated primary CD8+or CD4+T cells were activated in vitro for 18 hours with plate-bound anti-CD3 (1 pg / mL; Biolegend) and anti-CD28 (0.5 pg / mL; Biolegend) antibodies in Click’s media at 37°C and 5% CO2 incubator. Activated CD8+or CD4+T cells were ready for further experiments. A naive T cell control was maintained in Click’s media containing 10 ng / mL IL-7 (BioLegend).

[0150] Milk IgA, lactoferrin, IL-7, IL-lf level

[0151] After collecting milk samples from each group (five samples per group), 10 pL of each sample was used for IgA level detection by Mouse IgA ELISA Kit (Invitrogen, 88-50450-88), 10 pL of each sample was used for lactoferrin level detection by Mouse LTF / Lactoferrin ELISA Kit (Invitrogen, EEL103), 10 pL of each sample was used for IL-7 level detection by Mouse IL-7 ELISA Kit (Invitrogen, EMIL7), and 10 pL of each sample was used for IL- 10 level detection Attorney docket No. UCHI-43648.601

[0152] Client Ref. No. 24-T-025 by Mouse IL-1 beta ELISA Kit (Invitrogen, BMS6002), according to the manufacturer’s instructions.

[0153] Ex-vivo assay to assess the function of naive CD4+T cells from neonatal mice

[0154] TVA diet-fed and control (CON) diet-fed C57BL / 6J and Gpr43 / mouse lines were generated as described above. Neonatal offspring were harvested at postnatal day 10, and naive CD4+T cells were isolated for the following assays: Th polarization: Cells were seeded on plates precoated with anti-CD3 (1 pg / mL) and anti-CD28 (0.5 pg / mL) and incubated for 12 hours. Thereafter, Thl, Th2, or Th 17 differentiation cocktails were added, and cells were cultured for 48 hours. The frequency of polarized Th subsets was determined by flow cytometry. Early activation (CD69) assay: Naive CD4+T cells were stimulated on anti-CD3 / CD28-coated plates (same coating concentrations) for 6 hours, then stained for CD69 and analyzed by flow cytometry. Proliferation assay: Following a 12-hour activation on anti-CD3 / CD28-coated plates, cells were transferred to fresh medium containing 5 lU / mL human IL-2, 1 ng / mL mouse IL-7, and 5 ng / mL mouse IL-15, and cultured for an additional 48 hours. Cell expansion was assessed by counting or proliferation dye dilution.

[0155] In-vitro treatment for naive CD4+T cells

[0156] Naive CD4+T cells were isolated from neonatal (10-14 days old) or adult (8-12 weeks old) C57BL / 6J mice. Cells were treated with 10 ng / mL mouse IL-7 in the presence of either DMSO or 20 pM TVA for 24 hours. Following treatment, cells were stimulated on anti- CD3 / CD28-coated plates for 12 hours. Subsequently, Thl differentiation cocktails were added, and cells were cultured for an additional 48 hours. The frequency of Thl -polarized cells was then assessed by flow cytometry.

[0157] Similarly, naive CD4+T cells were isolated from neonatal (10-14 days old) Gpr43 / mice and treated with 10 ng / mL mouse IL-7 along with DMSO or 20 pM TVA for 24 hours, followed by anti-CD3 / CD28 stimulation for 12 hours. Thl differentiation cocktails were then applied, and cells were cultured for 48 hours. Thl polarization was analyzed by flow cytometry. To examine the role of cAMP-PKA signaling, naive CD4+T cells from neonatal (10-14 days old) C57BL / 6J mice were treated with 10 ng / mL IL-7 and either DMSO, 20 pM TVA, 200 nM PKA inhibitor H-89 dihydrochloride, or 1 pM 8-Bromo-cAMP for 24 hours. Cells were then Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 stimulated with anti-CD3 / CD28 for 12 hours, followed by Thl polarization for 48 hours. Thl cell frequency was assessed by flow cytometry.

[0158] To investigate transcriptional regulation, naive CD4+T cells from neonatal (10-14 days old) C57BL / 6J mice were treated with Accell siQc / (Horizon. E-044693-00-0020), Accell iCrebl (Horizon, E-040959-00-0020), or Accell control siRNA (Horizon, D-001910-10-05) according to the manufacturer’s protocol. After confirming knockdown efficiency, cells were treated with DMSO or 20 pM TVA for 24 hours, followed by 12-hour anti-CD3 / CD28 stimulation and 48-hour Thl differentiation. The frequency of Thl cells was analyzed by flow cytometry.

[0159] Electrophoretic Mobility Shift Assay (EMSA) for CTCF Activity

[0160] CTCF DNA-binding activity was assessed using the Pierce™ LightShift™ Chemiluminescent EMSA Kit (Thermo Fisher Scientific, cat# 20148) according to the manufacturer’s instructions. A biotin-labclcd double-stranded DNA oligonucleotide probe containing a known CTCF-binding motif was synthesized (Integrated DNA Technologies) with the following sequence: 5'-gtagtgacccCGgagccaCGtggagccaccttcagacccagagctgaagcaggagggtgacCGg-3' (SEQ ID NO: 1; biotin-dT at the 5' end of the reverse strand). For in vitro treatment assays, naive CD4+T cells were isolated from five 10-day-old C57BL / 6J mice per sample. Cells were treated with 10 ng / mL recombinant mouse IL-7 in the presence of DMSO (vehicle), 20 pM TVA, 200 nM PKA inhibitor H-89 dihydrochloride, or 1 pM 8-Bromo-cAMP for 24 hours. Nuclear proteins were then extracted using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, cat# 78833). For imprinting experiments in adult mice, naive CD4+T cells were isolated from TVA-to-CON and CON-to-CON adult mice (five mice per group). Nuclear extracts were prepared as described above. Equal amounts of nuclear protein (quantified by BCA assay) were incubated with the biotin-labeled DNA probe and subjected to non-denaturing polyacrylamide gel electrophoresis. DNA-protein complexes were transferred to a nylon membrane, detected by chemiluminescence, and quantified by densitometry to evaluate the ratio of CTCF-DNA complex / free probe to indicate CTCF activity. Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0161] Neonatal mouse model for influenza A virus challenge

[0162] C57BL / 6J neonatal mice from the control or TVA groups were challenged with Influenza A virus (H1N1 influenza A virus A / Puerto Rico / 8 / 1934; PR8). Briefly, 10-day-old neonatal mice from either the TVA or control diet groups were intranasally infected with Influenza virus PR8 (0.04 TCID50 / g in 7 pL saline). The pups were weighed and monitored daily for activity levels, respiratory rate, and maternal interaction. Neonates that were ignored by the mother, exhibited weight loss, or showed lack of movement were removed from the cage. The neonates were observed for two weeks for survival experiments, and the clinical score was rated daily (Score 0: No signs of disease; Active, well-groomed, feeding normally; Normal breathing pattern. Score 1: Slight ruffling of fur; Slight reduction in activity; Subtle weight loss (<5% expected body weight). Score 2: Noticeable fur ruffling; Reduced activity; less responsive to stimuli; Mild dehydration; Moderate weight loss (5-10% expected body weight). Score 3: Severe ruffling of fur and hunched posture; Marked reduction in movement and responsiveness; Labored breathing (tachypnea or dyspnea); Severe weight loss (10-20% expected body weight). Score 4: Unresponsive to external stimuli; Cyanosis or visibly labored breathing; Severe dehydration; Extreme weight loss (>20% expected body weight). Score 5: Death. Mice were also sacrificed on Day 4, day 7, or day 10 post-infection for evaluation of viral load, lung health, and immune responses. Lung samples were also collected on Day 7 post-infection for single-nuclei multi- omics analysis, including snRNA-seq and snATAC-seq (samples from 5 mice / group pooled).

[0163] Mouse lung infiltrating cell isolation

[0164] Neonatal C57BL / 6 mice challenged with Influenza A virus were euthanized, placed in a supine position, and secured with pins. The chest area was disinfected with 70% ethanol. Using sterile scissors, the thoracic cavity was carefully opened to expose the heart. A 23G needle was inserted into the right ventricle, and 10-15 mL of ice-cold PBS was slowly perfused to flush out circulating blood. Lungs were carefully dissected, rinsed in PBS to remove residual blood, and transferred to a Petri dish containing 5 mL of RPML1640 supplemented with 2% fetal bovine serum (FBS). Lung tissues were minced into small fragments using sterile scissors and transferred to a 15 mL conical tube containing 5 mL of digestion buffer (RPML1640 supplemented with 1 mg / mL Collagenase D and 100 U / mL DNase I). The tissue suspension was incubated at 37 °C for 30-40 minutes with gentle shaking every 5-10 minutes. Following Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 digestion, the cell suspension was passed through a 70 pm cell strainer into a 50 mL conical tube to obtain a single-cell suspension. The strainer was subsequently washed with an additional 5 mL of RPMI-1640 containing 2% FBS. Red blood cells were lysed by incubating the suspension with RBC lysis buffer for 2-3 minutes at room temperature. The cells were then washed with RPMI-1640 and centrifuged at 300 x g for 5 minutes at 4 °C. The resulting cell pellet was designated as lung-infiltrating cells and used for subsequent experiments.

[0165] Mouse lung IL-ip and TNF-a level

[0166] Lung tissues were collected from mice at 4, 7, 10 days post influenza A virus challenge, and rinsed briefly in cold PBS to remove blood contaminants. Tissues were weighed and homogenized in ice-cold PBS supplemented with a protease inhibitor cocktail (Roche, Complete Mini) using a bead mill homogenizer. Homogenates were centrifuged at 12,000 x g for 15 minutes at 4°C, and the supernatants were collected for analysis. IL- 10 and TNF-a levels in the lung homogenates were measured using a commercially available mouse IL- 10 ELISA kit (Biolegend, 432601) and TNF-a ELISA kit (Biolegend, 430901) according to the manufacturer’s instructions.

[0167] Mouse serum TNF-a level

[0168] Blood samples were collected from mice at 4, 7, 10 days post influenza A virus challenge, samples were centrifuged at 2,000 x g for 10 minutes at 4°C to isolate the serum. The supernatant (serum) was carefully collected and stored at -80°C until analysis. Serum TNF-a levels were quantified using a commercially available mouse TNF-a ELISA kit (Biolegend, 430901) according to the manufacturer’s instructions.

[0169] Single nuclei Multiome for spleen

[0170] Single nuclei Multiome library preparation, sequencing, and analysis were conducted by Singulomics Corporation. Single nuclei gene expression libraries and single nuclei ATAC libraries were constructed from nuclei isolated from cryop reserved mouse spleen cells on the lOx Genomics Chromium System using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle. Cryopreserved, viable single cell suspensions were thawed, washed, resuspended in cell culture media with 0.04% BSA, and counted. The cells were lysed Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 and permeabilized with Nonidet P40 Substitute, Digitonin, and Tween-20. The isolated nuclei were purified, centrifuged, and resuspended in Diluted Nuclei Buffer. The nuclei were then loaded into the Chromium Controller (lOx Genomics, Pleasanton, CA) to encapsulate single nuclei into droplet emulsions following the manufacturer’s recommendations. 6,000 nuclei were targeted to be captured per sample. Library preparation was performed according to the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide. Amplified cDNAs and the libraries were measured by Qubit dsDNA HS assay (Thermo Fisher Scientific, Wilmington, DE) and quality assessed by TapeStation (Agilent Technologies, Santa Clara, CA). Libraries were sequenced on a NovaSeq 6000 instrument (Illumina, San Diego, CA, United States), and reads were subsequently processed using lOx Genomics Cell Ranger ARC (v 2.0.2) analytical pipeline and mouse mm 10 (2020- A) reference. Dataset aggregation was performed using the cellranger-arc aggr function, normalizing for the total number of confidently mapped reads across libraries.

[0171] Single nuclei Multiome for lung

[0172] Single nuclei Multiome library preparation, sequencing, and analysis were conducted by Singulomics Corporation. Single nuclei gene expression libraries and single nuclei ATAC libraries were constructed from nuclei isolated from flash-frozen mouse lung tissue samples on the lOx Genomics Chromium System using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle. Flash-frozen mouse lung tissue samples were homogenized, and lysed and permeabilized with Nonidet P40 Substitute, Digitonin, and Tween- 20. The isolated nuclei were purified, centrifuged, and resuspended in Diluted Nuclei Buffer. The nuclei were then loaded into the Chromium Controller (lOx Genomics, Pleasanton, CA) to encapsulate single nuclei into droplet emulsions following the manufacturer’s recommendations. 7,000 nuclei were targeted to be captured per sample. Library preparation was performed according to the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide. Amplified cDNAs and the libraries were measured by Qubit dsDNA HS assay (Thermo Fisher Scientific, Wilmington, DE) and quality assessed by TapeStation (Agilent Technologies, Santa Clara, CA). Libraries were sequenced on a NovaSeq 6000 instrument (Illumina, San Diego, CA, United States), and reads were subsequently processed using lOx Genomics Cell Ranger ARC (v 2.0.2) analytical pipeline and mouse mmlO (2020- A) reference. Dataset Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 aggregation was performed using the cell ranger-arc aggr function, normalizing for the total number of confidently mapped reads across libraries.

[0173] Single nuclei Multiome data analysis

[0174] All data were generated using the lOx Chromium Single Cell Multiome platform. For each sample, the analysis input is the Cell Ranger ARC output, then we applied Seurat v5.1.0(47) and Signac v 1.13.0 to create a multi-omic Seurat object with paired gene expression and DNA accessibility profiles for each sample. The lOx multiome workflow encompasses quality control (QC), dimensionality reduction, clustering, and UMAP visualization across RNA, ATAC, and a joint neighbor graph. This graph represents both gene expression and DNA accessibility measurements via weighted nearest neighbor (WNN) methods. The workflow also includes cell type annotations, gene activity assessment, peak calling using MACS2, and the identification and visualization of condition- specific candidate cis-regulatory elements (cCREs), differentially active motifs, and key regulators. For each sample, we conducted QC, dimensionality reduction, clustering, and cell type annotations, followed by the integration of all samples for downstream analysis. Then, transcription factor “activity” was predicted based on the presence of binding motifs within DA chromatin regions using chromVAR, and the positional weight matrix (PWM) was obtained from the JASPAR2020 database. Paired data provides a unique opportunity to identify transcription factors (TFs) that satisfy multiple criteria, helping to narrow down the list of putative regulators to the most likely candidates. Experiments were conducted during development of embodiments herein to identify TFs whose expression is enriched in multiple cell types in the RNA measurements and with enriched accessibility for their motifs in the ATAC measurements. To do this, the “presto” package was used to perform fast differential expression in gene expression data and chromVAR motif accessibilities, presto calculates an “AUC” statistic, which reflects the power of each gene (or motif) to serve as a marker of cell type. A maximum AUC value of 1 indicates a perfect marker. Since the AUC statistic is on the same scale for both genes and motifs, the average of the AUC values was calculated from the two tests and use this to rank TFs for each cell type. Then, the TF expression (RNA level), gene activity (ATAC level), and motif activity (chromVAR) of the key regulators were visualized. Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0175] TCRa KO mouse model

[0176] 8-week-old TCRa KO male and female mice were fed with the TVA-enriched diet or control diet for two weeks before pairing them for breeding. Their offspring were kept on TVA- enriched diet or control diet and inbred to continue this line. After the control diet and TVA diet mouse lines stabilized (2-3 generations), the neonatal pups of each group were used for further analysis. 10-day-old neonatal mice from either the TVA or control diet groups were intranasally infected with Influenza virus PR8 (0.01 TCID50 / g in 7 pL saline). The pups were weighed and monitored daily for activity levels, respiratory rate, and maternal interaction. Neonates that were ignored by the mother, exhibited weight loss, or showed lack of movement were removed from the cage. The neonates were observed for two weeks for survival experiments.

[0177] (ipr43 mouse model

[0178] 8-week-old Gpr43~'~ male and female mice were fed with the TVA-enriched diet or control diet for two weeks before setting pairing them for breeding. Their offspring were kept on TVA-enriched diet or control diet and inbred to continue this line. After the control diet and TVA diet mouse lines stabilized (2-3 generations), the neonatal pups of each group were used for further analysis. 10-day-old neonatal mice from either the TVA or control diet groups were intranasally infected with Influenza virus PR8 (0.04 TCID50 / g in 7 pL saline). The pups were weighed and monitored daily for activity levels, respiratory rate, and maternal interaction. Neonates that were ignored by the mother, exhibited weight loss, or showed lack of movement were removed from the cage. The neonates were observed for two weeks for survival experiments.

[0179] Tbx r ' mouse model

[0180] 8-week-old Tbxll’ ’ male and female mice were fed with the TVA-enriched diet or control diet for two weeks before setting pairing them for breeding. Their offspring were kept on TVA-enriched diet or control diet and inbred to continue this line. After the control diet and TVA diet mouse lines stabilized (2-3 generations), the neonatal pups of each group were used for further analysis. 10-day-old neonatal mice from either the TVA or control diet groups were intranasally infected with Influenza virus PR8 (0.02 TCID50 / g in 7 pL saline). The pups were weighed and monitored daily for activity levels, respiratory rate, and maternal interaction. Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0181] Neonates that were ignored by the mother, exhibited weight loss, or showed lack of movement were removed from the cage. The neonates were observed for two weeks for survival experiments.

[0182] Human milk fatty acid profiling

[0183] The twenty-two preterm infants (with an Estimated Gestational Age of 24-33 weeks) were divided into two groups based on whether they were diagnosed with Bronchopulmonary Dysplasia (BPD), which was determined by the need for supplemental oxygen at 36 weeks postmenstrual age. The paired milk from the infants’ mothers or donors was used for fatty acid profiling. Approximately 200 pL of milk sample was spiked with the 25 pL of 100 pM ofl 3C>- trans vaccenic acid and extracted with methods modified from Matyash et al. In brief, the milk sample was mixed with the 800 pL ice-cold methanol containing 0.1 mg / mL butylated hydroxy toluene and sonicated for 15 minutes followed by the addition of 2400 pL of methyl- tcrt-butyl ether and sonicated for 30 minutes at room temperature. The phase separation was induced by adding 800 pL of water, sonication for 15 minutes, and centrifuge at 18,000g for 15 minutes. The upper 2000 pL of the organic layer was collected in a scintillation vial, dried down using the Genevac vacuum dryer, and stored at -20°C until analyses. On the day of the LC-MS analysis, samples were re-suspended in 80 pL of 3 / 2 / 1 isopropanol / acetonitrile / water, sonicated for 2 minutes, thermomixer for 10 minutes at 15°C and supernatant was transferred to LC-MS vial after spinning them down at 18,000g for 20 minutes at 15 °C. The fatty acids were separated on Cortecs T3 (2.1x100mm, 1.6 micron, part # 186008499 Waters Corporation) column connected to a Vanquish Horizon UHPLC system and IQ-X tribrid mass spectrometers. The column temperature, injection volume, and flow rate were 40°C, 5 pL, and 0.3mL / minute, respectively. The mobile phase A (MPA) was 60 / 40 acetonitrile / water, 10 mM ammonium formate+0.1% formic acid, and MPB was 89.1 / 9.9 / 0.99 isopropanol / acetonitrile / water, 10 mM ammonium formate +0.1% formic acid. The chromatographic gradient was 0 minute: 5%B, 1.2 minute: 5%B, 5 minutes: 50%B, 9.00 minute: 50%B, 13.00 minute: 70%B, 15.5 minute: 70%B, 17.00 minute: 100%B, 20.00 minute: 100%B, 20.2 minute: 5%B, 28.00 minute: 5%B. MSI parameters were as follows: spray voltage: 2800 V negative, sheath gas: 40, auxiliary gas: 10, sweep gas: 1, ion transfer tube temperature: 300°C, vaporizer temperature: 350°C, orbitrap resolution: 60K, scan range(m / z): 120-500, RF lens (%): 60, automatic gain control (AGC) Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 target: 100%, and a maxIT of 118 milliseconds (ms). The fatty acids were identified by matching their retention time to the respective commercially available reference standard.

[0184] Mouse-fostering model

[0185] 8-week-old male and female C57BL / 6 and FVB / NJ mice were fed either a control diet or a 0.1% TVA-supplemented diet for three weeks. Following this, the mice were bred within their respective control or TVA groups. Once both C57BL / 6 and FVB / NJ mice gave birth on the same day, 1-day-old pups from C57BL / 6 mice on the control diet were randomly and equally assigned to either control or TVA-fed FVB / NJ foster mothers. Similarly, 1-day-old pups from C57BL / 6 mice on the TVA diet were randomly and equally assigned to either control or TVA-fed FVB / NJ foster mothers. At 10 days old, the four groups of pups were challenged with influenza A virus. We monitored survival and clinical scores for two weeks, and on day 7 post-infection, lung tissue was collected for virus load and immune cell flow cytometry analysis, which includes cell type markers CD45, CD4, CD8, CD3, TCR-dclta, NK1.1, CDl lb, Ly6G, F4 / 80, Ly6C, MHCII, CD11c, B220; CD4 and CD 8 activation or influenza virus -specific react markers CD49d, CD1 la, CD44, CD69, Tetramer NP331-325, Tetramer PA224-233.

[0186] Mouse model of immune imprinting effects

[0187] C57BL / 6J mice were fed either a trans-vaccenic acid (TVA) diet or a control (CON) diet as described above to generate TVA and CON diet-fed mouse lines. Offspring were nursed by their mothers until weaning at 3 weeks of age, during which time they received breastmilk derived from either the TVA or CON diet-fed dams. After weaning, all offspring were placed on the CON diet and maintained until adulthood (6-10 weeks of age). Peripheral blood samples were collected from offspring at both 3 weeks (weaning) and adulthood (6-10 weeks) to measure circulating TVA levels. Naive CD4+T cells were isolated from each group to assess CTCF transcriptional activity and Thl polarization capacity. In a separate experiment, adult offspring (6-10 weeks old) were intranasally challenged with influenza virus PR8 at a dose of 0.4 TCIDso / g body weight in 20 pL sterile saline. Mice were monitored daily to assess survival.

[0188] Mouse serum cytokine array

[0189] 2-week-old mouse serum samples from each group were used for the cytokine profiling Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 by MILLIPLEX® Mouse Cytokine / Chemokine Magnetic Bead Panel - Immunology Multiplex Assay Kit (Millipore Sigma, MCYTOMAG-70K) according to the manufacturer’s instructions.

[0190] Blood spot card cytokine detection

[0191] Six 3.2-mm (1 / 8-inch) diameter disks were punched from dried blood-spots. The disks were eluted together in 180 L of PBS containing a "Complete protease inhibitor cocktail without EDTA" (Pierce), including 0.5 g / L sodium azide, by agitating at room temperature for 2 hours. Then the mixture was centrifuged at 1000g for 10 minutes. The supernatant was collected for cytokine assay using related cytokine detection ELISA kit as the manufacturer’s instructions.

[0192] Cell staining and flow cytometry

[0193] Mouse primary cells isolated from spleens, thymus, and lungs were stained with fluorescent antibodies and analyzed by flow cytometry.

[0194] For experiments with livc / dcad criteria, cells were first stained with Fixable Viability Dyes (FVD) (Thermo Fisher Scientific) according to the manufacturer’s instructions. Subsequent surface marker staining was performed in the Flow Cytometry Staining Buffer (Thermo Fisher Scientific). Intracellular staining for flow panels containing nuclear proteins was performed using the eBioscience FoxP3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions. For intracellular staining of cytoplasmic proteins, the Fixation / Permeabilization Solution Kit (BD Biosciences) was used according to the manufacturer’s instructions. For PE-conjugated influenza A NP3ii-325 / I-Ab tetramer staining, incubate for 120 minutes at 37°C in the dark. For influenza A PA224-233 / Db tetramer staining, incubate for 120 minutes at 4°C. For phospho antibody staining, cells were incubated with FVD (cell viability dye) for 15 minutes at room temperature in a tube, re-suspended with 200 pL prewarmed IxBD Phosflow Lyse / Fix buffer directly into the tube, and incubated at 37°C for 10-15 minutes, followed by centrifugation at 300xg for 5 minutes. The resulting cell pellets were washed once with FACS buffer, permeabilized with 200 p.L of BD Phosflow Perm Buffer III for 45 minutes on ice and centrifuged at 300xg for 5 minutes. The cell pellets were washed again with FACS buffer, centrifuged at 300xg for 5 minutes, and incubated with antibodies in FACS buffer for 45 minutes- 1 hour at room temperature.

[0195] After gating live CD45+cells, CD4+T cells are gated as CD3+CD4+CD8“, CD8+T cells Attorney docket No. UCHI-43648.601

[0196] Client Ref. No. 24-T-025 are gated as CD3+CD8+CD4", NK cells are gated as CD3-NK1.1+, B cells are gated as B220+CD19+, macrophages are gated as F4 / 80+CDl lb+Ly6G“, monocytes are gated as CDl lb+Ly6C+Ly6G-F4 / 80low, DCs are gated as CDllc+MHC-II+, neutrophils are gated as CDl lb+Ly6G+Ly6Cint. For thymus, after gating live CD45+cells, DN (Double-Negative) are gated as CD4-CD8“, DP (Double-Positive) are gated as CD4+CD8+, CD4 Single-Positive (SP) are gated as CD4+CD8“, CD8 Single-Positive (SP) are gated as CD4“CD8+.

[0197] Data was collected on BD LSR-Fortessa 4-15 flow cytometer, Cytek Aurora, or Attune NxT 4- 14 and analyzed using FlowJo vl0.4.

[0198] Antibodies

[0199] Mouse PerCP / Cyanine5.5 anti-Ki-67 Antibody (Biolegend, Cat#652423; Clone#16A8; RRID:AB_2629530, 1:200); PE anti-TCFl (TCF7) Antibody (Biolegend, Cat#655207; Clone#7Fl 1A10; RRID:AB_2728491, 1:200); Human / mouse / rat FITC anti-CD278 (ICOS) Antibody (Biolcgcnd, Cat#313505; Clonc#C398.4A; RRID:AB_416329, 1:200); Human / mousc FITC anti-Granzyme B Recombinant Antibody (Biolegend, Cat#372205; Clone#QA16A02; RRID:AB_2687029, 1:200); Mouse PE / Cyanine5 anti-CD69 Antibody (Biolegend, Cat#104509; Clone#H1.2F3; RRID:AB_313112, 1:200); Mouse PE / Cyanine5 anti-CD4 Antibody (Biolegend, Cat#100409; Clone#GK1.5; RRID:AB 312694, 1:200); Mouse Brilliant Violet 421™ anti-IL-2 Antibody (Biolegend, Cat#503825; Clone#JES6-5H4; RRID:AB_10895901, 1:200); Mouse APC anti-CD45.2 Antibody (Biolegend, Cat#109813; Clone#104; RRID:AB_389210, 1:200); Mouse APC anti-IFN-y Antibody (Biolegend, Cat#5O581O; Clone#XMG1.2; RRID:AB_315404, 1:200); Human / mouse PE / Cyanine7 anti-Granzyme B Recombinant Antibody (Biolegend, Cat#372213; Clone#QA16A02; RRID:AB_2728380, 1:200); Mouse PerCP / Cyanine5.5 anti- TNF-a Antibody (Biolegend, Cat#506321; Clone#MP6-XT22; RRID:AB_961435, 1:200); Mouse Brilliant Violet 711™ anti-CD8a Antibody (Biolegend, Cat#100747; Clone#53-6.7; RRID:AB_11219594, 1:200); Mouse Brilliant Violet 421™ anti-FOXP3 Antibody (Biolegend, Cat#126419; Clone#MF-14; RRID:AB_2565933, 1:200); Mouse APC anti-CD3 Antibody (Biolegend, Cat#100235; Clone#17A2; RRID:AB_2561455, 1:200); Mouse FITC anti-F4 / 80 Recombinant Antibody (Biolegend, Cat#157309; Clone#QA17A29; RRID:AB_2876535, 1:200); Mouse APC anti-Ly-6G (Grl) Antibody (Biolegend, Cat#127613; Clone#lA8;

[0200] RRID:AB_1877163, 1:200); Mouse / human APC anti-CDl lb Antibody (Biolegend, Cat#101211; Attorney docket No. UCHI-43648.601

[0201] Client Ref. No. 24-T-025

[0202] Clone#Ml / 70; RRID: AB_312794, 1:200); Mouse PerCP anti-CDl lc Antibody (Biolegend, Cat#l 17325; Clone#N418; RRID: AB_893236, 1:200); Mouse Mouse / human PE anti-Ki-67 Antibody (Biolegend, Cat#151210; Clone#l lF6; RRID:AB_2716008, 1:200); APC Phospho- CREB (Seri 33) Recombinant Rabbit Monoclonal Antibody (Thermo Fisher Scientific, Cat#MA5-36992; Clone#CREBS133-4Dl l; RRID:AB_2896927, 1:200); Rabbit PE Active Caspase-3 (Thermo Fisher Scientific, Cat#BDB561011; Clone#C92-605 ; RRID:AB_2033931, 1:200); Rabbit PE Phospho-Statl (Tyr701) Recombinant Monoclonal Antibody (Thermo Fisher Scientific, Cat#MA5-37039; Clone#StatlY701-3E6; RRID:AB_2896974, 1:200); GPR43 Polyclonal Antibody (Thermo Fisher Scientific, Cat#PA5-111780; Clone#N / A;

[0203] RRID:AB_2857189, 1:500); Mouse APC-cy7-B220 antibody (Biolegend, Cat#103223; Clone# RA3-6B2; RRID: AB_313006); Mouse BV711-CD19 antibody (Biolegend, Cat#115555; Clone# 6D5; RRID: AB_2565970); Mouse PE-CD69 antibody (Biolegend, Cat#104507; Clone# H1.2F3; RRID: AB_313110); Mouse BV421-CD62L antibody (Biolegend, Cat#104435; Clone# MEL-14; RRID: AB_10900082); Mouse FITC-CD25 antibody (Biolcgcnd, Cat#101907; Clone# 3C7; RRID: AB_961210); Mouse PE-cy7-CDl la antibody (Biolegend, Cat#153107; Clone# 121 / 7; RRID: AB_2716056); Mouse FITC-CD49d antibody (Biolegend, Cat#103605; Clone# Rl-2; RRID: AB_313036); Mouse BV421-NK1.1 antibody (Biolegend, Cat# 156537; Clone# S17016D; RRID: AB_3083138); Mouse PE-CD115 antibody (Biolegend, Cat# 135505; Clone# AFS98; RRID: AB_1937254); Mouse BV421-CD103 antibody (Biolegend, Cat# 121421; Clone# 2E7; RRID: AB_10900074); Mouse FITC-CD80 antibody (Biolegend, Cat# 104705; Clone# 16-10A1; RRID: AB.313126); Mouse FITC-CD86 antibody (Biolegend, Cat# 159219; Clone# A17199A; RRID: AB_3106043); PE-conjugated influenza A NP311-325 / 1- Ab tetramer (NIH tetramer core facility); PE-conjugated influenza A PA224-233 / Db tetramer (NIH tetramer core facility); Purified anti-Keratin 5 Antibody (Biolegend, Cat# 905503; Clone# Polyl9055; RRID: AB_2734679); Anti-Prosurfactant Protein C (proSP-C) Antibody (Millipore Cat# AB3786, RRID:AB_91588); Spark UV 387-Ly6G (Biolegend, Cat# 127677; Clone# 1A8; RRID: AB_2924466); BUV737-IL7R (BD Biosciences, Cat# 612841; RRID: AB_2870163); BV421-CD62L (Biolegend, Cat# 104435; Clone# MEL-14; RRID: AB_10900082); Pacific Blue-CD45 (Biolegend, Cat# 157211; Clone# SI 8OO9F; RRID: AB_2876534); BV510-TER119 (Biolegend, Cat# 116237; Clone# TER- 119; RRID: AB_2561661); BV605-Scal (Biolegend, Cat# 108133; Clone# D7; RRID: AB_2562275); BV650-CDl lb, (Biolegend, Cat# 101239; Attorney docket No. UCHI-43648.601

[0204] Client Ref. No. 24-T-025

[0205] Clone# MI / 70; RRID: AB_11125575); BV711-CD115 (Biolegend, Cat# 135515; Clone# AFS98; RRID: AB_2562679); BV785-CD150 (Biolegend, Cat# 115937; Clone# TC15-12F12.2; RRID: AB_2565962); FITC-CD62L (Biolegend, Cat# 161211; Clone# W1802 ID; RRID: AB_2941440); SB550-B220 (Biolegend, Cat# 103265; Clone# RA3-6B2; RRID: AB_2819793); PerCP-e710-CD135 (eBioscience, Cat# 46-1351-82; RRID:AB_10733393); RB780-Streptavidin (BD Biosciences, Cat# 570517); CD105 Biotin (Biolegend, Cat# 120403; Clone# MJ7 / 18;

[0206] RRID: AB_961059); PE-ESAM (Biolegend, Cat# 136203; Clone# 1G8 / ESAM; RRID: AB_1953300); PE-Dazzle594-CD48 (Biolegend, Cat# 103437; Clone# HM48-1; RRID: AB_2650822); PE-Cy5-CD 16 / 32 (Biolegend, Cat# 156617; Clone# S17011E; RRID: AB_2894425); PE-Cy7-CD41 (Biolegend, Cat# 133915; Clone# MWReg30; RRID: AB_11125173); APC-cKit (Biolegend, Cat# 105811; Clone# 2B8; RRID: AB.313220); Spark NIR685-CD3 (Biolegend, Cat# 100261; Clone# 17A2; RRID: AB_2832259); AF700-CD27 (Biolegend, Cat# 124239; Clone# LG.3A10; RRID: AB.2810382); APC-Cy7-Ly6C (Biolegend, Cat# 128025; Clone# HK1.4; RRID: AB_10643867); BUV805-CD127 (Invitrogen, Cat# 368- 1271-82; RRID: AB_2896132); e450-CD4 (Invitrogen, Cat# 48-0041-82; RRID:

[0207] AB_10718983); BV650-CD44 (Biolegend, Cat# 103049; Clone# IM7; RRID: AB_2562600); BV711-CXCR5 (Biolegend, Cat# 145529; Clone# L138D7; RRID: AB_2734207); BV785- CD25 (Biolegend, Cat# 102051; Clone# PC61; RRID: AB_2564131); FITC-TCRb (Biolegend, Cat# 109205; Clone# H57-597; RRID: AB_313428); PE-TCRyS (Biolegend, Cat# 118107; Clone# GL3; RRID: AB_313831); PE-Cy7-cKit (Biolegend, Cat# 135111; Clone# ACK2; RRID: AB_2131136); APC-ICOS (Biolegend, Cat# 313509; Clone# C398.4A; RRID: AB_416333); APC-e780-CD8 (Invitrogen, Cat# 47-0087-42; RRID: AB_2016684).

[0208] Milk microbiome 16S sequencing

[0209] After collecting milk samples from each group (three samples per group), 50 pL of each sample was mixed with 150 pL of DNA / RNA Shield™ (ZYMO Research, R1100-250) and subsequently sent to Zymo Research (Irvine, CA) for microbiome 16S rRNA sequencing. In brief, The ZymoBIOMICS®-96 MagBead DNA Kit (Zymo Research) was used to extract DNA using an automated platform. Bacterial 16S ribosomal RNA gene-targeted sequencing was performed using the Quick- 16S™ NGS Library Prep Kit (Zymo Research). The bacterial 16S primers amplified the V3-V4 region of the 16S rRNA gene. The sequencing library was prepared Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025 using an innovative library preparation process in which PCR reactions were performed in realtime PCR machines to control cycles and, therefore, limit PCR chimera formation. The final PCR products were quantified with qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned with the Select-a-Size DNA Clean & Concentrator™ (Zymo Research), then quantified with TapeStation® (Agilent Technologies) and Qubit® (Thermo Fisher Scientific). The ZymoBIOMICS® Microbial Community Standard (Zymo Research) was used as a positive control for each DNA extraction, if performed. The final library was sequenced on Illumina® MiSeq™ with a v3 reagent kit (600 cycles). The sequencing was performed with 10% PhiX spike-in. For Bioinformatics Analysis, unique amplicon sequences variants were inferred from raw reads using the DADA2 pipeline. Potential sequencing errors and chimeric sequences were also removed with the Dada2 pipeline. Chimeric sequences were also removed with the DADA2 pipeline. Taxonomy assignment was performed using Uclust from Qiime v.1.9.1 with the Zymo Research Database, a 16S database that is internally designed and curated, as reference. Composition visualization, alpha-diversity, and beta-diversity analyses were performed with Qiime v.1.9.1.

[0210] Quantitative real-time PCR (RT-PCR)

[0211] Total RNA was extracted with TRIzol Reagent (Invitrogen) and then used for synthesizing the first strand cDNA with PrimeScript™ 1st strand cDNA Synthesis Kit (Takara) according to the manufacturer’s instructions. Quantitative RT-PCR was conducted with iTaq Universal SYBR Green Supermix (Bio-Rad).

[0212] RNA interference (RNAi) with Accell siRNA

[0213] Neonatal mouse primary T cells were isolated and cultured in replete media (RPMI 1640 medium or Click’s medium containing 10% FBS, 55 pM 2-mercaptoethanol, 2 mM glutamine, penicillin / streptomycin, and either PHA, CD3 or IL-2) for 24 hours, followed by incubation with Accell delivery mix (Accell siRNA Delivery Media (Horizon Discovery) with 1 pM siRNA, 20 lU / mL IL-2 and 1% FBS) for 72 hours. Cells were collected for subsequent function analysis as well as depletion efficiency validation using RT-PCR. Attorney docket No. UCHI-43648.601

[0214] Client Ref. No. 24-T-025

[0215] Quantification and statistical analysis

[0216] Statistical analyses were performed using GraphPad Prism 9. Data figures are representative of at least three independent experiments or two independent experiments. All attempts at replication were successful.

[0217] REFERENCES

[0218] The following references are herein incorporated by reference in their entireties.

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[0220] 2. J. Uberos et al., Breast-feeding as protective factor against bronchopulmonary dysplasia in preterm infants. Br J Nutr 131, 1405-1412 (2024).

[0221] 3. L. Y. Kim, S. A. McGrath-Morrow, J. M. Collaco, Impact of breast milk on respiratory outcomes in infants with bronchopulmonary dysplasia. Pediatr Pulmonol 54, 313-318 (2019).

[0222] 4. X. Guo et al., Association between viral infection and bronchopulmonary dysplasia in preterm infants: a systematic review and meta-analysis. Eur J Pediatr 183, 2965-2981 (2024).

[0223] 5. A. Camacho-Morales et al., Breastfeeding Contributes to Physiological Immune Programming in the Newborn. Front Pediatr 9, 744104 (2021).

[0224] 6. S. Moossavi, K. Miliku, S. Sepehri, E. Khafipour, M. B. Azad, The Prebiotic and Probiotic Properties of Human Milk: Implications for Infant Immune Development and Pediatric Asthma. Front Pediatr 6, 197 (2018).

[0225] 7. O. Ballard, A. L. Morrow, Human milk composition: nutrients and bioactive factors. Pediatr Clin North Am 60, 49-74 (2013).

[0226] 8. D. K. Dror, L. H. Allen, Overview of Nutrients in Human Milk. Adv Nutr 9, 278S-294S (2018).

[0227] 9. I. Petersohn et al., Maternal diet and human milk composition: an updated systematic review. Front Nutr 10, 1320560 (2023).

[0228] 10. D. Ramiro-Cortijo et al., Breast Milk Lipids and Fatty Acids in Regulating Neonatal Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0229] Intestinal Development and Protecting against Intestinal Injury. Nutrients 12, (2020). D. Jamiol-Milc, E. Stachowska, T. Janus, A. Barcz, D. Chlubek, [Trans fatty acids (elaidic and vaccenic) in the human milk]. Pomeranian J Life Sci 61, 58-63 (2015). L. Te Morenga, J. M. Montez, Health effects of saturated and trans-fatty acid intake in children and adolescents: Systematic review and meta-analysis. PLoS One 12, e0186672 (2017). M. Sommerfeld, Trans unsaturated fatty acids in natural products and processed foods. Prog Lipid Res 22, 221-233 (1983). A. M. Turpeinen et al., Bioconversion of vaccenic acid to conjugated linoleic acid in humans. Am J Clin Nutr 76, 504-510 (2002). J. E. Santora, D. L. Palmquist, K. L. Roehrig, Trans-vaccenic acid is desaturated to conjugated linoleic acid in mice. J Nutr 130, 208-215 (2000). H. Fan et al., Trans-vaccenic acid reprograms CD8(+) T cells and anti-tumour immunity. Nature 623, 1034-1043 (2023). S. L. Swain, K. K. McKinstry, T. M. Strutt, Expanding roles for CD4(+) T cells in immunity to viruses. Nat Rev Immunol 12, 136-148 (2012). H. Zaghouani, C. M. Hoeman, B. Adkins, Neonatal immunity: faulty T-helpers and the shortcomings of dendritic cells. Trends Immunol 30, 585-591 (2009). S. Sattler, N. Rosenthal, The neonate versus adult mammalian immune system in cardiac repair and regeneration. Biochim Biophys Acta 1863, 1813-1821 (2016). B. D. Rudd, Neonatal T Cells: A Reinterpretation. Annu Rev Immunol 38, 229-247 (2020). C. T. Ong, V. G. Corces, CTCF: an architectural protein bridging genome topology and function. Nature Reviews Genetics 15, 234-246 (2014). M. Sekimata et al., CCCTC-Binding Factor and the Transcription Factor T-bet Orchestrate T Helper 1 Cell-Specific Structure and Function at the Interferon-' Locus. Immunity 31, 551-564 (2009). D. F. Smee, M. von Itzstein, B. Bhatt, E. B. Tarbet, Exacerbation of influenza vims infections in mice by intranasal treatments and implications for evaluation of antiviral drugs. Antimicrob Agents Chemother 56, 6328-6333 (2012). M. Fernanda de Mello Costa, A. I. Weiner, A. E. Vaughan, Basal-like Progenitor Cells: A Attorney docket No. UCHI-43648.601 Client Ref. No. 24-T-025

[0230] Review of Dysplastic Alveolar Regeneration and Remodeling in Lung Repair. Stem Cell Reports 15, 1015-1025 (2020).

[0231] 25. A. Schimpl et al., IL-2 and autoimmune disease. Cytokine Growth Factor Rev 13, 369- 378 (2002).

[0232] 26. U. Salimi, K. Dummula, M. H. Tucker, C. S. Dela Cruz, V. Sampath, Postnatal Sepsis and Bronchopulmonary Dysplasia in Premature Infants: Mechanistic Insights into "New BPD". Am J Respir Cell Mol Biol 66, 137-145 (2022).

[0233] 27. C. P. Speer, Inflammation and bronchopulmonary dysplasia: a continuing story. Semin Fetal Neonatal Med 11, 354-362 (2006).

[0234] 28. C. P. Speer, Pulmonary inflammation and bronchopulmonary dysplasia. J Perinatal 26 Suppl 1, S57-62; discussion S63-54 (2006).

[0235] 29. M. G. Constantinides, Y. Belkaid, Early-life imprinting of unconventional T cells and tissue homeostasis. Science 374, eabf'0095 (2021).

[0236] 30. E. C. Scmmcs ct al., Understanding Early-Life Adaptive Immunity to Guide Interventions for Pediatric Health. Front Immunol 11, 595297 (2020).

[0237] 31. D. L. Hickman, M. P. Swan, Effects of age of pups and removal of existing litter on pup survival during cross-fostering between multiparous outbred mice. J Am Assoc Lab Anim Sci 50, 641-646 (2011).

[0238] 32. I. L. Lines, S. Hoskins, M. Hollifield, L. S. Cauley, B. A. Garvy, The migration of T cells in response to influenza virus is altered in neonatal mice. J Immunol 185, 2980-2988 (2010).

[0239] 33. E. E. West et al., Loss of CD4+ T cell-intrinsic arginase 1 accelerates Thl response kinetics and reduces lung pathology during influenza infection. Immunity 56, 2036-+ (2023).

[0240] 34. H. M. Lazear, T. J. Nice, M. S. Diamond, Interferon-X: Immune Functions at Barrier Surfaces and Beyond. Immunity 43, 15-28 (2015).

[0241] 35. B. Adkins, C. Leclerc, S. Marshall-Clarke, Neonatal adaptive immunity comes of age. Nature Reviews Immunology 4, 553-564 (2004).

[0242] 36. K. H. Restori, B. T. Srinivasa, B. J. Ward, E. D. Fixman, Neonatal immunity, Respiratory virus infections, and the Development of Asthma. Frontiers in Immunology 9, (2018).

[0243] 37. O. Levy, Innate immunity of the newborn: basic mechanisms and clinical correlates. Attorney docket No. UCHI-43648.601

[0244] Client Ref. No. 24-T-025

[0245] Nature Reviews Immunology 7, 379-390 (2007). Y. Wang et al., Trans- 11 vaccenic acid dietary supplementation induces hypolipidemic effects in JCR:LA-cp rats. J Nutr 138, 2117-2122 (2008). S. K. Gebauer et al., Effects of ruminant trans fatty acids on cardiovascular disease and cancer: a comprehensive review of epidemiological, clinical, and mechanistic studies.

[0246] Adv Nutr 2, 332-354 (2011). I. G. Pranger, F. A. J. Muskiet, I. P. Kema, C. Singh-Povel, S. J. L. Bakker, Potential Biomarkers for Fat from Dairy and Fish and Their Association with Cardiovascular Risk Factors: Cross-sectional Data from the LifeLines Biobank and Cohort Study. Nutrients 11, (2019).

Claims

Attorney docket No. UCHI-43648.601Client Ref. No. 24-T-025CLAIMS1. A prenatal and / or postnatal supplement or formulation comprising trans-vaccenic acid (TVA), cis-l l,14-Eicosadienoic acid (EDA), nervonic acid (NVA), and / or a derivative thereof.

2. The prenatal and / or postnatal supplement or formulation of claim 1, further comprising one or more of folate, calcium, vitamin A, riboflavin, iodine, DHA, omega-3 fatty acids (e.g., docosahexaenoic acid (DHA), Eicosapentaenoic acid (EPA)), omega-6 fatty acids (e.g., Arachidonic acid (ARA)), pyridoxine, iron, choline, niacin, thiamin, magnesium, selenium, vitamin D, vitamin C, vitamin B12, vitamin E, vitamin K, iodine, amino acids, protein hydrolysates, non-digestible oligosaccharides, probiotics, synbiotics, chromium, fluoride, taurine, nucleotides, phospholipids, triacylglycerols, palmitic acid, L-camitine, inositol, and zinc.

3. The prenatal and / or postnatal supplement or formulation of claim 1, wherein the supplement is formulated for oral administration to a pregnant or lactating subject.

4. The prenatal and / or postnatal supplement or formulation of claim 1, wherein the supplement comprises TVA.

5. The prenatal and / or postnatal supplement or formulation of claim 1, wherein the supplement comprises a TVA derivative.

6. The prenatal and / or postnatal supplement or formulation of claim 5, wherein the TVA derivative is selected from Cl 8:1 trans- 10-octadecenoic acid, C18:l trans- 12-octadecenoic acid, C18:2 trans-11, cis-13, C17:l trans-9-heptadecenoic acid, C20:l trans- 13-eicosenoic acid, 11- methyl TVA, 12-hydroxy TVA, 11-phenyl TVA, and 11-amino TVA.

7. The prenatal and / or postnatal supplement or formulation of claim 1, wherein the supplement comprises TVD.

8. The prenatal and / or postnatal supplement or formulation of claim 1, wherein theAttorney docket No. UCHI-43648.601Client Ref. No. 24-T-025 supplement comprises an EDA derivative.

9. The prenatal and / or postnatal supplement or formulation of claim 8, wherein the EDA derivative is selected from C20:2 cis-12,15, C20:2 cis-9,12, C20:3 cis-8, 11,14, C18:2 cis-9,12, C22:2 cis-13,16, 13-methyl EDA, 14-hydroxy EDA, EDA with ethyl group at CIO, and EDA with phenyl group at C13.

10. The prenatal and / or postnatal supplement or formulation of claim 1, wherein the supplement comprises NVA.

11. The prenatal and / or postnatal supplement or formulation of claim 1, wherein the supplement comprises an NVA derivative.

12. The prenatal and / or postnatal supplement or formulation of claim 11, wherein the NVA derivative is selected from C24:l cis-13, C24:l cis-17, C24:2 cis- 15, 18, C22:l cis-13-docosenoic acid, C26:l cis-17-hexacosenoic acid, 15-methyl NVA, 16-hydroxy NVA, 15-amino NVA, 15- phenyl NVA, and NVA with propyl group at C12.

13. A method of enriching breast milk with TVA, EDA, NVA, and / or a derivative thereof comprising administering a prenatal and / or postnatal supplement of claim 1 to a pregnant or nursing subject.

14. A method of enhancing immune development and / or immune response in a newborn, infant, or pediatric subject comprising administering breast milk or infant formula or follow-on formula (FOF) comprising TVA, EDA, NVA, and / or a derivative thereof to the subject.

15. A method of enhancing immune development and / or immune response in a newborn or infant or pediatric subject comprising (a) administering a supplement comprising TVA, EDA, NVA, and / or a derivative thereof to a pregnant or lactating subject, and (b) feeding the newborn or infant or pediatric subject with breast milk from the pregnant or lactating subject.Attorney docket No. UCHI-43648.601Client Ref. No. 24-T-02516. An infant or baby formula, formulation, or supplement comprising trans-vaccenic acid (TVA), cis-11,14-Eicosadienoic acid (EDA), nervonic acid (NVA), and / or a derivative thereof.

17. The infant or baby formula, formulation, or supplement of claim 16, further comprising whey, casein, whey protein isolate, skim milk powder, vegetable oils, animal milk fats, milk oligosaccharides, iron, docosahexaenoic acid (DHA), arachidonic acid (ARA), folate, amino acids, and / or taurine.

18. The infant or baby formula, formulation, or supplement of claim 16, wherein the formula is formulated for oral administration to a newborn or infant or pediatric subject.

19. The infant or baby formula, formulation, or supplement of claim 16, comprising TVA.

20. The infant or baby formula, formulation, or supplement of claim 16, comprising a TVA derivative.

21. The infant or baby formula, formulation, or supplement of claim 20, wherein the TVA derivative is selected from Cl 8:1 trans- 10-octadecenoic acid, C18:l trans- 12-octadecenoic acid, C18:2 trans-11, cis-13, C17:l trans-9-heptadecenoic acid, C20:l trans- 13-eicosenoic acid, 11- methyl TVA, 12-hydroxy TVA, 11-phenyl TVA, and 11-amino TVA.

22. The infant or baby formula, formulation, or supplement of claim 16, comprising EDA.

23. The infant or baby formula, formulation, or supplement of claim 16, comprising an EDA derivative.

24. The infant or baby formula, formulation, or supplement of claim 23, wherein the EDA derivative is selected from C20:2 cis-12,15, C20:2 cis-9,12, C20:3 cis-8, 11,14, C18:2 cis-9,12, C22:2 cis-13, 16, 13-methyl EDA, 14-hydroxy EDA, EDA with ethyl group at CIO, and EDA with phenyl group at C13.Attorney docket No. UCHI-43648.601Client Ref. No. 24-T-02525. The infant or baby formula, formulation, or supplement of claim 16, comprising NVA.

26. The infant or baby formula, formulation, or supplement of claim 16, comprising an NVA derivative.

27. The infant or baby formula, formulation, or supplement of claim 26, wherein the NVA derivative is selected from C24:l cis-13, C24:l cis-17, C24:2 cis- 15, 18, C22:l cis-13-docosenoic acid, C26:l cis-17-hexacosenoic acid, 15-methyl NVA, 16-hydroxy NVA, 15-amino NVA, 15- phenyl NVA, and NVA with propyl group at C12.

28. Use of an effective dose of trans-vaccenic acid (TVA), cis-ll,14-Eicosadienoic acid (EDA), nervonic acid (NVA), and / or a derivative thereof for enhancing immune development and / or immune response in a newborn or infant or pediatric subject.

29. Use of an effective dose of trans-vaccenic acid (TVA), cis-11,14-Eicosadienoic acid (EDA), nervonic acid (NVA), and / or a derivative thereof in the manufacture of a supplement or formulation for administration to a pregnant or lactating subject.

30. Use of an effective dose of trans-vaccenic acid (TVA), cis-11,14-Eicosadienoic acid (EDA), nervonic acid (NVA), and / or a derivative thereof in the manufacture of a infant or baby formula, formulation, or supplement for administration to a newborn or infant or pediatric subject.

31. A method of treating, preventing, reducing the likelihood, or reducing the severity of an infection in a subject in need thereof comprising administering an effective amount of trans- vaccenic acid (TVA), cis-11,14-Eicosadienoic acid (EDA), nervonic acid (NVA), and / or a derivative thereof to the newborn or infant or pediatric subject.

32. A formulation, composition or kit comprising a trans-vaccenic acid (TVA), cis-11,14- Eicosadienoic acid (EDA), nervonic acid (NVA), and / or a TVA, EDA, or NVA derivative for the prevention and / or treatment of disease in a newborn or infant or pediatric subject.Attorney docket No. UCHI-43648.601Client Ref. No. 24-T-02533. A formulation, composition or kit comprising a trans-vaccenic acid (TV A), cis-11,14- Eicosadienoic acid (EDA), nervonic acid (NVA), and / or a TVA, EDA, or NVA derivative and an additional therapeutic or prophylactic agent for the prevention and / or treatment of disease in a newborn or infant or pediatric subject.

34. The formulation, composition or kit of claim 32 or 33, wherein the active TVA, NVA, EDA and / or a TVA, EDA, or NVA derivative can enhance spleen development and splenic CD4+T-cells in a newborn or infant or pediatric subject.

35. The formulation, composition or kit of claim 32 or 33, wherein the active TVA, NVA, EDA and / or a TVA, EDA, or NVA derivative can enhance thymus development and thymic CD4+CD8+ T cells in a newborn or infant or pediatric subject.

36. The formulation, composition or kit of claim 32 or 33, wherein the active TVA, NVA, EDA and / or a TVA, EDA, or NVA derivative can enhance CD8+ T cell activity and / or can enhance immunity.

37. The formulation, composition or kit of claim 32 or 33, wherein the active TVA, NVA, EDA and / or a TVA, EDA, or NVA derivative can prevent and / or treat infection in a newborn or infant or pediatric subject.

38. The formulation, composition or kit of claim 32 or 33, wherein the active TVA, NVA, EDA and / or a TVA, EDA, or NVA derivative can prevent and / or treat viral infection in a newborn or infant or pediatric subject.

39. The formulation, composition or kit of claim 32 or 33, wherein the active TVA, NVA, EDA and / or a TVA, EDA, or NVA derivative can prevent and / or treat bacterial infection in a newborn or infant or pediatric subject.

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