Charles Arama

Analysis of the 5q31-q33 Locus Shows an Association between IL13-1055C/T IL-13-591A/G Polymorphisms and Schistosoma haematobium Infections

Millions of humans are exposed to schistosome infections, which cause severe kidney and liver disease and 280,000 deaths annually. Th2-mediated immunity is critical to human defenses against this pathogen and susceptibility to infection is controlled by a major genetic locus that includes IL4, IL5, and IL13 genes. These observations led us to evaluate whether certain polymorphisms in IL4, IL5, or IL13 determine schistosome infection. The study was performed in two Dogon villages where Schistosoma haematobium is endemic. Schistosome infections were evaluated by counting eggs and measuring worm Ags in urine. Genetic polymorphisms were determined by restriction enzyme analysis or by primer extension and denaturing high-performance liquid chromatography analysis. Associations were tested using family-based association tests and logistical regression analysis. The alleles IL13-1055C (p = 0.05) and IL13-591A (p = 0.01) are shown, by family-based association test, to be preferentially transmitted to children with the 10% highest infections. A logistic regression analysis that included IL13-1055 G/G, G/T and T/T genotypes, age, gender, and village of residency, applied to the whole study population, showed that subjects bearing the IL13-1055T/T genotype were on average much less infected than individuals with other genotypes. Previous studies on asthma indicated that the IL13-1055T allele increased gene transcription, which is in agreement with the fact that this cytokine enhances resistance to infection by schistosome in humans.

Effects of concomitant Schistosoma haematobium infection on the serum cytokine levels elicited by acute Plasmodium falciparum malaria infection in Malian children.

ABSTRACT Polyparasitism is common in the developing world, and interactions that alter disease severity may occur. We previously demonstrated that infection with Schistosoma hematobium was associated with protection against Plasmodium falciparum infection in children who were 4 to 8 years old. In this study, we determined whether underlying helminth infections affected the cytokine responses to acute falciparum malaria. A total of 338 schistosomiasis-positive [Sch(+)] children who were 4 to 14 years old were matched by age, residence, and sex with 338 schistosomiasis-negative [Sch(−)] children and monitored for a malaria transmission season (25 weeks). Serologic cytokine levels were measured at the time of the first clinical malaria episode and in children who did not contract malaria. Elevated background levels of interleukin-6 (IL-6) (37.1 pg/ml versus 10.9 pg/ml [P = 0.04]), IL-4 (27.7 pg/ml versus 6.9 pg/ml [P = 0.02]), IL-10 (18.2 pg/ml versus 7.2 pg/ml [P < 0.001]), and gamma interferon (18.2 pg/ml versus 4.7 pg/ml [P = 0.006]) were noted in Sch(+) children compared to Sch(−) children without malaria. IL-6 and IL-10 levels were elevated in association with acute malaria, but the levels appeared to be blunted in Sch(+) children compared to Sch(−) children who were 4 to 8 years old (for IL-6, 96.2 pg/ml versus 137.2 pg/ml [P = 0.08]; for IL-10, 195.9 pg/ml versus 282.2 pg/ml [P = 0.06]). The level of IL-10 was similarly lower in Sch(+) children than in Sch(−) children who were 9 to 14 years old (91.2 pg/ml versus 141.2 pg/ml [P = 0.03]). IL-4 levels were inversely correlated with the time until the first malaria infection in both the Sch(+) children (P < 0.001) and the Sch(−) children (P < 0.001) who were 4 to 8 years old. We postulate that the Th2-enriched environment induced by schistosomiasis protects against malaria and alters the cytokine milieu during an actual infection.

Early interferon-gamma response against Plasmodium falciparum correlates with interethnic differences in susceptibility to parasitemia between sympatric Fulani and Dogon in Mali.

INTRODUCTION Interethnic differences in susceptibility to malaria provide a unique opportunity to explore immunological correlates of protection. The Fulani of Sahelian Africa are known for their reduced susceptibility to Plasmodium falciparum, compared with surrounding tribes, yet the immunology underlying this is still poorly understood. METHODS AND RESULTS Here, we show that mononuclear cells from Fulani elicit >10-fold stronger interferon (IFN)-gamma production following a 24-h in vitro coincubation with asexual parasites than cells from sympatric Dogon. This response appears to be specific for P. falciparum among a panel of other human pathogens and is independent of the lower number of regulatory T cell counts present in Fulani. IFN-gamma responses in both tribes were inversely correlated with peripheral parasite density as quantified by nucleic acid sequenced-based amplification, but responses of Fulani remained significantly stronger than those of Dogon after adjustment for concurrent parasitemia, suggesting that hard-wired immunological differences underlie the observed protection. CONCLUSIONS These results underscore the value of early IFN-gamma responses to P. falciparum as a correlate of anti-parasite immunity, not only in this setting but also in the wider context of malaria, and support the development of malaria vaccines aimed at inducing such responses.

Interethnic Differences in Antigen-Presenting Cell Activation and TLR Responses in Malian Children during Plasmodium falciparum Malaria

The Fulani ethnic group from West Africa is relatively better protected against Plasmodium falciparum malaria as compared to other sympatric ethnic groups, such as the Dogon. However, the mechanisms behind this lower susceptibility to malaria are largely unknown, particularly those concerning innate immunity. Antigen-presenting cells (APCs), and in particular dendritic cells (DCs) are important components of the innate and adaptive immune systems. Therefore, in this study we investigated whether APCs obtained from Fulani and Dogon children exhibited differences in terms of activation status and toll-like receptor (TLR) responses during malaria infection. Lower frequency and increased activation was observed in circulating plasmacytoid DCs and BDCA-3+ myeloid DCs of infected Fulani as compared to their uninfected counterparts. Conversely, a higher frequency and reduced activation was observed in the same DC subsets obtained from peripheral blood of P. falciparum-infected Dogon children as compared to their uninfected peers. Moreover, infected individuals of both ethnic groups exhibited higher percentages of both classical and inflammatory monocytes that were less activated as compared to their non-infected counterparts. In line with APC impairment during malaria infection, TLR4, TLR7 and TLR9 responses were strongly inhibited by P. falciparum infection in Dogon children, while no such TLR inhibition was observed in the Fulani children. Strikingly, the TLR-induced IFN-γ release was completely abolished in the Dogon undergoing infection while no difference was seen within infected and non-infected Fulani. Thus, P. falciparum infection is associated with altered activation status of important APC subsets and strongly inhibited TLR responses in peripheral blood of Dogon children. In contrast, P. falciparum induces DC activation and does not affect the innate response to specific TLR ligands in Fulani children. These findings suggest that DCs and TLR signalling may be of importance for the protective immunity against malaria observed in the Fulani.

Changes in the levels of cytokines, chemokines and malaria-specific antibodies in response to Plasmodium falciparum infection in children living in sympatry in Mali

BackgroundThe Fulani are known to be less susceptible to Plasmodium falciparum malaria as reflected by lower parasitaemia and fewer clinical symptoms than other sympatric ethnic groups. So far most studies in these groups have been performed on adults, which is why little is known about these responses in children. This study was designed to provide more information on this gap.MethodsCirculating inflammatory factors and antibody levels in children from the Fulani and Dogon ethnic groups were measured. The inflammatory cytokines; interleukin (IL)-1beta, IL-6, IL-8, IL-10, IL-12p70, tumor necrosis factor (TNF) and the chemokines; regulated on activation normal T cell expressed and secreted (RANTES), monokine-induced by IFN-gamma (MIG), monocyte chemotactic protein (MCP)-1 and IFN-gamma-inducible protein (IP)-10 were measured by cytometric bead arrays. The levels of interferon (IFN)-alpha, IFN-gamma and malaria-specific antibodies; immunoglobulin (Ig) G, IgM and IgG subclasses (IgG1-IgG4) were measured by ELISA.ResultsThe results revealed that the Fulani children had higher levels of all tested cytokines compared to the Dogon, in particular IFN-gamma, a cytokine known to be involved in parasite clearance. Out of all the tested chemokines, only MCP-1 was increased in the Fulani compared to the Dogon. When dividing the children into infected and uninfected individuals, infected Dogon had significantly lower levels of RANTES compared to their uninfected peers, and significantly higher levels of MIG and IP-10 as well as MCP-1, although the latter did not reach statistical significance. In contrast, such patterns were not seen in the infected Fulani children and their chemokine levels remained unchanged upon infection compared to uninfected counterparts. Furthermore, the Fulani also had higher titres of malaria-specific IgG and IgM as well as IgG1-3 subclasses compared to the Dogon.ConclusionsTaken together, this study demonstrates, in accordance with previous work, that Fulani children mount a stronger inflammatory and antibody response against P. falciparum parasites compared to the Dogon and that these differences are evident already at an early age. The inflammatory responses in the Fulani were not influenced by an active infection which could explain why less clinical symptoms are seen in this group.

Reduced T regulatory cell response during acute Plasmodium falciparum infection in Malian children co-infected with Schistosoma haematobium.

Background Regulatory T cells (Tregs) suppress host immune responses and participate in immune homeostasis. In co-infection, secondary parasite infections may disrupt the immunologic responses induced by a pre-existing parasitic infection. We previously demonstrated that schistosomiasis-positive (SP) Malian children, aged 4–8 years, are protected against the acquisition of malaria compared to matched schistosomiasis-negative (SN) children. Methods and Findings To determine if Tregs contribute to this protection, we performed immunologic and Treg depletion in vitro studies using PBMC acquired from children with and without S. haematobium infection followed longitudinally for the acquisition of malaria. Levels of Tregs were lower in children with dual infections compared to children with malaria alone (0.49 versus 1.37%, respectively, P = 0.004) but were similar months later, during a period with negligible malaria transmission. The increased levels of Tregs in SN subjects were associated with suppressed serum Th1 cytokine levels, as well as elevated parasitemia compared to co-infected counterparts. Conclusions These results suggest that lower levels of Tregs in helminth-infected children correlate with altered circulating cytokine and parasitologic results which may play a partial role in mediating protection against falciparum malaria.

A recombinant Bacille Calmette-Guerin construct expressing the Plasmodium falciparum circumsporozoite protein enhances dendritic cell activation and primes for circumsporozoite-specific memory cells in BALB/c mice

A protective malaria vaccine may induce both high levels of neutralising antibodies and strong T-cell responses. The Plasmodium falciparum circumsporozoite protein (CSp) is a leading pre-erythrocytic vaccine candidate. CSp is a week immunogen per se, but Mycobacterium bovis Bacille Calmette-Guérin (BCG) has excellent adjuvant activity and has been utilized as a vector to deliver heterologous vaccine candidate antigens. It is safe in immunocompetent individuals and inexpensive to produce. We assessed in vitro and in vivo a recombinant BCG-expressing CSp (BCG-CS) as malaria vaccine candidate. Immunisation of BALB/c mice with BCG-CS augmented numbers of dendritic cells (DCs) in draining lymph nodes and in the spleen. The activation markers MHC-class-II, CD40, CD80 and CD86 on DCs were significantly upregulated by BCG-CS as compared to wild-type BCG (wt-BCG). In vitro stimulation of bone marrow-derived DCs and macrophages with BCG-CS induced IL-12 and TNF-α production. BCG-CS induced higher phagocytic activity in macrophages as compared to wt-BCG. Immunogenicity studies show that BCG-CS induced CS-specific antibodies and IFN-γ-producing memory cells. In conclusion, BCG-CS is highly efficient in activating antigen-presenting cells (APCs) for priming of adaptive immunity. Implications for the rational design of novel vaccines against malaria and TB, the two major devastating poverty-related diseases, are discussed.

Antigen-Specific B Memory Cell Responses to Plasmodium falciparum Malaria Antigens and Schistosoma haematobium Antigens in Co-Infected Malian Children

Polyparasitism is common in the developing world. We have previously demonstrated that schistosomiasis-positive (SP) Malian children have age-dependent protection from malaria compared to matched schistosomiasis-negative (SN) children. Evidence of durable immunologic memory to malaria antigens is conflicting, particularly in young children and the effect of concomitant schistomiasis upon acquisition of memory is unknown. We examined antigen-specific B memory cell (MBC) frequencies (expressed as percentage of total number of IgG-secreting cells) in 84 Malian children aged 4–14 to malaria blood-stage antigens, apical membrane antigen 1 (AMA-1) and merozoite surface protein 1 (MSP-1) and to schistosomal antigens, Soluble Worm Antigenic Preparation (SWAP) and Schistosoma Egg Antigen (SEA), at a time point during the malaria transmission season and a follow-up dry season visit. We demonstrate, for the first time, MBC responses to S. haematobium antigens in Malian children with urinary egg excretion and provide evidence of seasonal acquisition of immunologic memory, age-associated differences in MBC acquisition, and correlation with circulating S. haematobium antibody. Moreover, the presence of a parasitic co-infection resulted in older children, aged 9–14 years, with underlying S. haematobium infection having significantly more MBC response to malaria antigens (AMA1 and MSP1) than their age-matched SN counterparts. We conclude that detectable MBC response can be measured against both malaria and schistosomal antigens and that the presence of S. haematobium may be associated with enhanced MBC induction in an age-specific manner.

Protection of Malian children from clinical malaria is associated with recognition of multiple antigens

BackgroundNaturally acquired immunity to clinical malaria is thought to be mainly antibody-mediated, but reports on antigen targets are contradictory. Recognition of multiple antigens may be crucial for protection. In this study, the magnitude of antibody responses and their temporal stability was assessed for a panel of malaria antigens in relation to protection against clinical Plasmodium falciparum malaria.MethodsMalian children aged two to 14 years were enrolled in a longitudinal study and followed up by passive and active case detection for seven months. Plasma was collected at enrolment and at the beginning, in the middle and after the end of the transmission season. Antibody titres to the P. falciparum-antigens apical membrane protein (AMA)-1, merozoite surface protein (MSP)-119, MSP-3, glutamine-rich protein (GLURP-R0) and circumsporozoite antigen (CSP) were assessed by enzyme-linked immunosorbent assay (ELISA) for 99 children with plasma available at all time points. Parasite carriage was determined by microscopy and nested PCR.ResultsAntibody titres to all antigens, except MSP-119, and the number of antigens recognized increased with age. After malaria exposure, antibody titres increased in children that had low titres at baseline, but decreased in those with high baseline responses. No significant differences were found between antibody titers for individual antigens between children remaining symptomatic or asymptomatic after exposure, after adjustment for age. Instead, children remaining asymptomatic following parasite exposure had a broader repertoire of antigen recognition.ConclusionsThe present study provides immune-epidemiological evidence from a limited cohort of Malian children that strong recognition of multiple antigens, rather than antibody titres for individual antigens, is associated with protection from clinical malaria.

Heterologous prime-boost regimen adenovector 35-circumsporozoite protein vaccine/recombinant Bacillus Calmette-Guérin expressing the Plasmodium falciparum circumsporozoite induces enhanced long-term memory immunity in BALB/c mice

BACKGROUND Sustained antibody levels are a hallmark of immunity against many pathogens, and induction of long-term durable antibody titers is an essential feature of effective vaccines. Heterologous prime-boost approaches with vectors are optimal strategies to improve a broad and prolonged immunogenicity of malaria vaccines. RESULTS In this study, we demonstrate that the heterologous prime-boost regimen Ad35-CS/BCG-CS induces stronger immune responses by enhancing type 1 cellular producing-cells with high levels of CSp-specific IFN-γ and cytophilic IgG2a antibodies as compared to a homologous BCG-CS and a heterologous BCG-CS/CSp prime-boost regimen. Moreover, the heterologous prime-boost regimen elicits the highest level of LLPC-mediated immune responses. CONCLUSION The increased IFN-γ-producing cell responses induced by the combination of Ad35-CS/BCG-CS and sustained type 1 antibody profile together with high levels of LLPCs may be essential for the development of long-term protective immunity against liver-stage parasites.