g Protection from infections in early life relies extensively on innate immunity, but it is unknown whether and how maternal infections modulate infants' innate immune responses, thereby altering susceptibility to infections. Plasmodium falciparum causes pregnancy-associated malaria (PAM), and epidemiological studies have shown that PAM enhances infants' susceptibility to infection with P. falciparum. We investigated how PAM-mediated exposures in utero affect innate immune responses and their relationship with infection in infancy. In a prospective study of mothers and their babies in Benin, we investigated changes in Toll-like receptor (TLR)-mediated cytokine responses related to P. falciparum infections. Whole-blood samples from 134 infants at birth and at 3, 6, and 12 months of age were stimulated with agonists specific for TLR3, TLR4, TLR7/8, and TLR9. TLRmediated interleukin 6 (IL-6) and IL-10 production was robust at birth and then stabilized, whereas tumor necrosis factor alpha (TNF-␣) and gamma interferon (IFN-␥) responses were weak at birth and then increased. In multivariate analyses, maternal P. falciparum infections at delivery were associated with significantly higher TLR3-mediated IL-6 and IL-10 responses in the first 3 months of life (P < 0.05) and with significantly higher TLR3-, TLR7/8-, and TLR9-mediated TNF-␣ responses between 6 and 12 months of age (P < 0.05). Prospective analyses showed that higher TLR3-and TLR7/8-mediated IL-10 responses at birth were associated with a significantly higher risk of P. falciparum infection in infancy (P < 0.05). Neonatal and infant intracellular TLRmediated cytokine responses are conditioned by in utero exposure through PAM late in pregnancy. Enhanced TLR-mediated IL-10 responses at birth are associated with an increased risk of P. falciparum infection, suggesting a compromised ability to combat infection in early life.

Maternal parasitoses modulate fetal immune development, manifesting as altered cellular immunological activity in cord blood that may be linked to enhanced susceptibility to infections in early life. Plasmodium falciparum typifies such infections, with distinct placental infection-related changes in cord blood exemplified by expanded populations of parasite antigen-specific regulatory T cells. Here we addressed whether such early-onset cellular immunological alterations persist through infancy. Specifically, in order to assess the potential impacts of P. falciparum infections either during pregnancy or during infancy, we quantified lymphocyte subsets in cord blood and in infants' peripheral blood during the first year of life. The principal age-related changes observed, independent of infection status, concerned decreases in the frequencies of CD4+, NKdim and NKT cells, whilst CD8+, Treg and Teff cells' frequencies increased from birth to 12 months of age. P. falciparum infections present at delivery, but not those earlier in gestation, were associated with increased frequencies of Treg and CD8+ T cells but fewer CD4+ and NKT cells during infancy, thus accentuating the observed age-related patterns. Overall, P. falciparum infections arising during infancy were associated with a reversal of the trends associated with maternal infection i.e. with more CD4+ cells, with fewer Treg and CD8+ cells. We conclude that maternal P. falciparum infection at delivery has significant and, in some cases, year-long effects on the composition of infants' peripheral blood lymphocyte populations. Those effects are superimposed on separate and independent age- as well as infant infection-related alterations that, respectively, either match or run counter to them.

IntroductionNeonatal sepsis outreaches all causes of neonatal mortality worldwide and remains a major societal burden in low and middle income countries. In addition to limited resources, endemic morbidities, such as malaria and prematurity, predispose neonates and infants to invasive infection by altering neonatal immune response to pathogens. Nevertheless, thoughtful epidemiological, diagnostic and immunological evaluation of neonatal sepsis and the impact of gestational malaria have never been performed.Methods and analysisA prospective longitudinal multicentre follow-up of 580 infants from birth to 3 months of age in urban and suburban Benin will be performed. At delivery, and every other week, all children will be examined and clinically evaluated for occurrence of sepsis. At delivery, cord blood systematic analysis of selected plasma and transcriptomic biomarkers (procalcitonin, interleukin (IL)-6, IL-10, IP10, CD74 and CX3CR1) associated with sepsis pathophysiology will be evaluated in all live births as well as during the follow-up, and when sepsis will be suspected. In addition, whole blood response to selected innate stimuli and extensive peripheral blood mononuclear cells phenotypic characterisation will be performed. Reference intervals specific to sub-Saharan neonates will be determined from this cohort and biomarkers performances for neonatal sepsis diagnosis and prognosis tested.Ethics and disseminationEthical approval has been obtained from the Comité d’Ethique de la Recherche – Institut des Sciences Biomédicales Appliquées (CER-ISBA 85 - 5 April 2016, extended on 3 February 2017). Results will be disseminated through international presentations at scientific meetings and publications in peer-reviewed journals.Trial registration numberClinicalTrials.gov registration number: NCT03780712.

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