Ontogeny of plasma cytokine and chemokine concentrations across the first week of human life.

INTRODUCTION/BACKGROUND & AIMS: Early life is marked by distinct and rapidly evolving immunity and increased susceptibility to infection. The vulnerability of the newborn reflects development of a complex immune system in the face of rapidly changing demands during the transition to extra-uterine life. Cytokines and chemokines contribute to this dynamic immune signaling network and can be altered by many factors, such as infection. Newborns undergo dynamic changes important to health and disease, yet there is limited information regarding human neonatal plasma cytokine and chemokine concentrations over the first week of life. The few available studies are limited by small sample size, cross-sectional study design, or focus on perturbed host states like severe infection or prematurity. To characterize immune ontogeny among healthy full-term newborns, we assessed plasma cytokine and chemokine concentrations across the first week of life in a robust longitudinal cohort of healthy, full-term African newborns. METHODS: We analyzed a subgroup of a cohort of healthy newborns at the Medical Research Council Unit in The Gambia (West Africa; N = 608). Peripheral blood plasma was collected from all study participants at birth (day of life (DOL) 0) and at one follow-up time point at DOL 1, 3, or 7. Plasma cytokine and chemokine concentrations were measured by bead-based cytokine multiplex assay. Unsupervised clustering was used to identify patterns in plasma cytokine and chemokine ontogeny during early life. RESULTS: We observed an increase across the first week of life in plasma Th1 cytokines such as IFNγ and CXCL10 and a decrease in Th2 and anti-inflammatory cytokines such as IL-6 and IL-10, and chemokines such as CXCL8. In contrast, other cytokines and chemokines (e.g. IL-4 and CCL5, respectively) remained unchanged during the first week of life. This robust ontogenetic pattern did not appear to be affected by gestational age or sex. CONCLUSIONS: Ontogeny is a strong driver of newborn plasma-based levels of cytokines and chemokines throughout the first week of life with a rising IFNγ axis suggesting post-natal upregulation of host defense pathways. Our study will prove useful to the design and interpretation of future studies aimed at understanding the neonatal immune system during health and disease.

Human In vitro Modeling Identifies Adjuvant Combinations that Unlock Antigen Cross-presentation and Promote T-helper 1 Development in Newborns, Adults and Elders.

Adjuvants are vaccine components that can boost the type, magnitude, breadth, and durability of an immune response. We have previously demonstrated that certain adjuvant combinations can act synergistically to enhance and shape immunogenicity including promotion of Th1 and cytotoxic T-cell development. These combinations also promoted protective immunity in vulnerable populations such as newborns. In this study, we employed combined antigen-specific human in vitro models to identify adjuvant combinations that could synergistically promote the expansion of vaccine-specific CD4+ cells, induce cross-presentation on MHC class I, resulting in antigen-specific activation of CD8+ cells, and direct the balance of immune response to favor the production of Th1-promoting cytokines. A screen of 78 adjuvant combinations identified several T cell-potentiating adjuvant combinations. Remarkably, a combination of TLR9 and STING agonists (CpG + 2,3-cGAMP) promoted influenza-specific CD4+ and CD8+ T cell activation and selectively favored production of Th1-polarizing cytokines TNF and IL-12p70 over co-regulated cytokines IL-6 and IL-12p40, respectively. Phenotypic reprogramming towards cDC1-type dendritic cells by CpG + 2,3-cGAMP was also observed. Finally, we characterized the molecular mechanism of this adjuvant combination including the ability of 2,3-cGAMP to enhance DC expression of TLR9 and the dependency of antigen-presenting cell activation on the Sec22b protein important to endoplasmic reticulum-Golgi vesicle trafficking. The identification of the adjuvant combination CpG + 2,3-cGAMP may therefore prove key to the future development of vaccines against respiratory viral infections tailored for the functionally distinct immune systems of vulnerable populations such as older adults and newborns.

CAF08 adjuvant enables single dose protection against respiratory syncytial virus infection in murine newborns.

Respiratory syncytial virus is a leading cause of morbidity and mortality in children, due in part to their distinct immune system, characterized by impaired induction of Th 1 immunity. Here we show application of cationic adjuvant formulation CAF08, a liposomal vaccine formulation tailored to induce Th 1 immunity in early life via synergistic engagement of Toll-like Receptor 7/8 and the C-type lectin receptor Mincle. We apply quantitative phosphoproteomics to human dendritic cells and reveal a role for Protein Kinase C-δ for enhanced Th1 cytokine production in neonatal dendritic cells and identify signaling events resulting in antigen cross-presentation. In a murine in vivo model a single immunization at birth with CAF08-adjuvanted RSV pre-fusion antigen protects newborn mice from RSV infection by induction of antigen-specific CD8+ T-cells and Th1 cells. Overall, we describe a pediatric adjuvant formulation and characterize its mechanism of action providing a promising avenue for development of early life vaccines against RSV and other respiratory viral pathogens.

Human Blood Plasma Shapes Distinct Neonatal TLR-Mediated Dendritic Cell Activation via Expression of the MicroRNA Let-7g.

The newborn innate immune system is characterized as functionally distinct, resulting in impaired proinflammatory responses to many stimuli and a bias toward Th2 development. Although the magnitude of impairment can be partially overcome, for instance through activation of TLR7/8 in newborn dendritic cells, the newborn innate response remains distinct from that of adults. Using human in vitro modeling of newborn and adult dendritic cells, we investigated the role of extracellular and intracellular regulators in driving age-specific responses to TLR7/8 stimulation. MicroRNA expression profiling and plasma switch experiments identified Let-7g as a novel regulator of newborn innate immunity. Activation-induced expression of Let-7g in monocyte-derived dendritic cells (MoDCs) is driven by newborn plasma and reduces expression of costimulatory receptors CD86, MHC class I, and CCR7 and secretion of IFN-α and sCD40L. Conversely, an increase in secretion of the Th2-polarizing cytokine IL-12p40 is observed. Overexpression of Let-7g in adult MoDCs resulted in the same observations. Small interfering RNA-mediated ablation of Let-7g levels in newborn MoDCs resulted in an adult-like phenotype. In conclusion, this study reveals for the first time (to our knowledge) that age-specific differences in human plasma induce the microRNA Let-7g as a key mediator of the newborn innate immune phenotype. These observations shed new light on the mechanisms of immune ontogeny and may inform approaches to discover age-specific immunomodulators, such as adjuvants.

Plasma Adenosine Deaminase (ADA)-1 and -2 Demonstrate Robust Ontogeny Across the First Four Months of Human Life.

Background: Human adenosine deaminases (ADAs) modulate the immune response: ADA1 via metabolizing adenosine, a purine metabolite that inhibits pro-inflammatory and Th1 cytokine production, and the multi-functional ADA2, by enhancing T-cell proliferation and monocyte differentiation. Newborns are relatively deficient in ADA1 resulting in elevated plasma adenosine concentrations and a Th2/anti-inflammatory bias compared to adults. Despite the growing recognition of the role of ADAs in immune regulation, little is known about the ontogeny of ADA concentrations. Methods: In a subgroup of the EPIC002-study, clinical data and plasma samples were collected from 540 Gambian infants at four time-points: day of birth; first week of life; one month of age; and four months of age. Concentrations of total extracellular ADA, ADA1, and ADA2 were measured by chromogenic assay and evaluated in relation to clinical data. Plasma cytokines/chemokine were measured across the first week of life and correlated to ADA concentrations. Results: ADA2 demonstrated a steady rise across the first months of life, while ADA1 concentration significantly decreased 0.79-fold across the first week then increased 1.4-fold by four months of life. Males demonstrated significantly higher concentrations of ADA2 (1.1-fold) than females at four months; newborns with early-term (37 to <39 weeks) and late-term (≥41 weeks) gestational age demonstrated significantly higher ADA1 at birth (1.1-fold), and those born to mothers with advanced maternal age (≥35 years) had lower plasma concentrations of ADA2 at one month (0.93-fold). Plasma ADA1 concentrations were positively correlated with plasma CXCL8 during the first week of life, while ADA2 concentrations correlated positively with TNFα, IFNγ and CXCL10, and negatively with IL-6 and CXCL8. Conclusions: The ratio of plasma ADA2/ADA1 concentration increased during the first week of life, after which both ADA1 and ADA2 increased across the first four months of life suggesting a gradual development of Th1/Th2 balanced immunity. Furthermore, ADA1 and ADA2 were positively correlated with cytokines/chemokines during the first week of life. Overall, ADA isoforms demonstrate robust ontogeny in newborns and infants but further mechanistic studies are needed to clarify their roles in early life immune development and the correlations with sex, gestational age, and maternal age that were observed.