A Balanced Proinflammatory and Regulatory Cytokine Signature in Young African Children Is Associated With Lower Risk of Clinical Malaria.

BACKGROUND: The effect of timing of exposure to first Plasmodium falciparum infections during early childhood on the induction of innate and adaptive cytokine responses and their contribution to the development of clinical malaria immunity is not well established. METHODS: As part of a double-blind, randomized, placebo-controlled trial in Mozambique using monthly chemoprophylaxis with sulfadoxine-pyrimethamine plus artesunate to selectively control timing of malaria exposure during infancy, peripheral blood mononuclear cells collected from participants at age 2.5, 5.5, 10.5, 15, and 24 months were stimulated ex vivo with parasite schizont and erythrocyte lysates. Cytokine messenger RNA expressed in cell pellets and proteins secreted in supernatants were quantified by reverse-transcription quantitative polymerase chain reaction and multiplex flow cytometry, respectively. Children were followed up for clinical malaria from birth until 4 years of age. RESULTS: Higher proinflammatory (interleukin [IL] 1, IL-6, tumor necrosis factor) and regulatory (IL-10) cytokine concentrations during the second year of life were associated with reduced incidence of clinical malaria up to 4 years of age, adjusting by chemoprophylaxis and prior malaria exposure. Significantly lower concentrations of antigen-specific T-helper 1 (IL-2, IL-12, interferon-γ) and T-helper 2 (IL-4, IL-5) cytokines by 2 years of age were measured in children undergoing chemoprophylaxis compared to children receiving placebo (P < .03). CONCLUSIONS: Selective chemoprophylaxis altering early natural exposure to malaria blood stage antigens during infancy had a significant effect on T-helper lymphocyte cytokine production >1 year later. Importantly, a balanced proinflammatory and anti-inflammatory cytokine signature, probably by innate cells, around age 2 years was associated with protective clinical immunity during childhood. CLINICAL TRIALS REGISTRATION: NCT00231452.

High production of pro-inflammatory cytokines by maternal blood mononuclear cells is associated with reduced maternal malaria but increased cord blood infection.

BACKGROUND: Increased susceptibility to malaria during pregnancy is not completely understood. Cellular immune responses mediate both pathology and immunity but the effector responses involved in these processes have not been fully characterized. Maternal and fetal cytokine and chemokine responses to malaria at delivery, and their association with pregnancy and childhood outcomes, were investigated in 174 samples from a mother and child cohort from Mozambique. Peripheral and cord mononuclear cells were stimulated with Plasmodium falciparum lysate and secretion of IL-12p70, IFN-γ, IL-2, IL-10, IL-8, IL-6, IL-4, IL-5, IL-1ß, TNF, TNF-ß was quantified in culture supernatants by multiplex flow cytometry while cellular mRNA expression of IFN-γ, TNF, IL-2, IL-4, IL-6, IL-10 and IL-13 was measured by quantitative PCR. RESULTS: Higher concentrations of IL-6 and IL-1ß were associated with a reduced risk of P. falciparum infection in pregnant women (p < 0.049). Pro-inflammatory cytokines IL-6, IL-1ß and TNF strongly correlated among themselves (ρ > 0.5, p < 0.001). Higher production of IL-1ß was significantly associated with congenital malaria (p < 0.046) and excessive TNF was associated with peripheral infection and placental lesions (p < 0.044). CONCLUSIONS: Complex network of immuno-pathological cytokine mechanisms in the placental and utero environments showed a potential trade-off between positive and negative effects on mother and newborn susceptibility to infection.

IgM and IgG against Plasmodium falciparum lysate as surrogates of malaria exposure and protection during pregnancy.

BACKGROUND: Difficulties to disentangle the protective versus exposure role of anti-malarial antibodies hamper the identification of clinically-relevant immune targets. Here, factors affecting maternal IgG and IgMs against Plasmodium falciparum antigens, as well as their relationship with parasite infection and clinical outcomes, were assessed in mothers and their children. Antibody responses among 207 Mozambican pregnant women at delivery against MSP119, EBA175, AMA1, DBLα and parasite lysate (3D7, R29 and E8B parasite lines), as well as the surface of infected erythrocytes, were assessed by enzyme-linked immunosorbent assay and flow cytometry. The relationship between antibody levels, maternal infection and clinical outcomes was assessed by multivariate regression analysis. RESULTS: Placental infection was associated with an increase in maternal levels of IgGs and IgMs against a broad range of parasite antigens. The multivariate analysis including IgGs and IgMs showed that the newborn weight increased with increasing IgG levels against a parasite lysate, whereas the opposite association was found with IgMs. IgGs are markers of protection against poor pregnancy outcomes and IgMs of parasite exposure. CONCLUSIONS: Adjusting the analysis for the simultaneous effect of IgMs and IgGs can contribute to account for heterogeneous exposure to P. falciparum when assessing immune responses effective against malaria in pregnancy.

Impact of age of first exposure to Plasmodium falciparum on antibody responses to malaria in children: a randomized, controlled trial in Mozambique.

BACKGROUND: The impact of the age of first Plasmodium falciparum infection on the rate of acquisition of immunity to malaria and on the immune correlates of protection has proven difficult to elucidate. A randomized, double-blind, placebo-controlled trial using monthly chemoprophylaxis with sulphadoxine-pyrimethamine plus artesunate was conducted to modify the age of first P. falciparum erythrocytic exposure in infancy and assess antibodies and malaria risk over two years. METHODS: Participants (n = 349) were enrolled at birth to one of three groups: late exposure, early exposure and control group, and were followed up for malaria morbidity and immunological analyses at birth, 2.5, 5.5, 10.5, 15 and 24 months of age. Total IgG, IgG subclasses and IgM responses to MSP-1(19), AMA-1, and EBA-175 were measured by ELISA, and IgG against variant antigens on the surface of infected erythrocytes by flow cytometry. Factors affecting antibody responses in relation to chemoprophylaxis and malaria incidence were evaluated. RESULTS: Generally, antibody responses did not vary significantly between exposure groups except for levels of IgM to EBA-175, and seropositivity of IgG1 and IgG3 to MSP-1(19). Previous and current malaria infections were strongly associated with increased IgG against MSP-1(19), EBA-175 and AMA-1 (p < 0.0001). After adjusting for exposure, only higher levels of anti-EBA-175 IgG were significantly associated with reduced clinical malaria incidence (IRR 0.67, p = 0.0178). CONCLUSIONS: Overall, the age of first P. falciparum infection did not influence the magnitude and breadth of IgG responses, but previous exposure was critical for antibody acquisition. IgG responses to EBA-175 were the strongest correlate of protection against clinical malaria. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00231452.

The role of age and exposure to Plasmodium falciparum in the rate of acquisition of naturally acquired immunity: a randomized controlled trial.

BACKGROUND: The rate of acquisition of naturally acquired immunity (NAI) against malaria predominantly depends on transmission intensity and age, although disentangling the effects of these is difficult. We used chemoprophylaxis to selectively control exposure to P. falciparum during different periods in infancy and explore the effect of age in the build-up of NAI, measured as risk of clinical malaria. METHODS AND FINDINGS: A three-arm double-blind randomized placebo-controlled trial was conducted in 349 infants born to Mozambican HIV-negative women. The late exposure group (LEG) received monthly Sulfadoxine-Pyrimethamine (SP) plus Artesunate (AS) from 2.5-4.5 months of age and monthly placebo from 5.5-9.5 months; the early exposure group (EEG) received placebo from 2.5-4.5 months and SP+AS from 5.5-9.5 months; and the control group (CG) received placebo from 2.5-9.5 months. Active and passive case detection (PCD) were conducted from birth to 10.5 and 24 months respectively. The primary endpoint was time to first or only episode of malaria in the second year detected by PCD. The incidence of malaria during the second year was of 0.50, 0.51 and 0.35 episodes/PYAR in the LEG, EEG and CG respectively (p = 0.379 for the adjusted comparison of the 3 groups). The hazard ratio of the adjusted comparison between the LEG and the CG was 1.38 (0.83-2.28, p = 0.642) and that between the EEG and the CG was 1.35 (0.81-2.24, p = 0.743). CONCLUSIONS: After considerably interfering with exposure during the first year of life, there was a trend towards a higher risk of malaria in the second year in children who had received chemoprophylaxis, but there was no significant rebound. No evidence was found that the age of first exposure to malaria affects the rate of acquisition of NAI. Thus, the timing of administration of antimalarial interventions like malaria vaccines during infancy does not appear to be a critical determinant. TRIAL REGISTRATION: ClinicalTrials.gov NCT00231452.