High titers of IgG antibodies against Plasmodium falciparum serine repeat antigen 5 (SERA5) are associated with protection against severe malaria in Ugandan children

Plasmodium falciparum serine repeat antigen (SERA5) is a promising asexual blood stage malaria candidate vaccine. However, there is a paucity of information about natural immune responses to SERA5 in children from malaria-endemic regions. We undertook a hospital-based case-control study of severe malaria in Apac District, Northern Uganda, in children 6-59 months of age. The commonest symptoms observed in children with severe malaria (SM) were respiratory distress (53.4%) and prostration (40.4%) followed by circulatory collapse (7.4%), severe anemia (Hb < 5 g/dL, 7.0%), and seizures (2.6%). None of the SM children had impaired consciousness, coma, or cerebral malaria. We measured serum IgG antibodies using a recombinant construct of SERA5 (SE36) in enzyme-linked immunosorbent assays. High titers of IgG anti-SE36 were associated with protection against severe malaria in children under 5 years old.     

Genetic diversity of the msp-1 locus and symptomatic malaria in south-west Nigeria

Genetic characteristics of Plasmodium falciparum may play a role in the clinical severity of malaria infection. We have studied the association between diversity at the merozoite surface protein-1 (msp-1) locus and the severity of disease in childhood malaria in Ibadan, south-west Nigeria. Two hundred and twenty-three children (median age of 34.5 months) presenting with malaria were enrolled into the study. They comprised 53 children with asymptomatic malaria (ASM), 101 with acute uncomplicated malaria (UM) and 69 with severe malaria (SM). Genotyping of the msp-1 locus was by polymerase chain reaction. The distribution of msp-1 alleles was significantly different between the three groups. Asymptomatic malaria samples had a higher median number of alleles than the other two groups. The type of msp-1 allele detected was significantly associated with the clinical category of malaria. The absence of K1 alleles was associated with a three-fold increase risk of UM and a four-fold increased risk of SM when compared with asymptomatic malaria. The absence of MAD20 alleles was associated with a five-fold increase risk of UM and an eight-fold increase of SM. We have found an association between the msp-1 locus of P. falciparum and clinical severity of malaria in a sample of Nigerian children. Our findings show that the presence of the K1 and MAD20 alleles was significantly associated with ASM and consequently a reduced risk of developing the symptomatic disease.     

Differential induction of immunoglobulin G to Plasmodium falciparum variant surface antigens during the transmission season in Daraweesh, Sudan

BACKGROUND: The acquisition of immunoglobulin (Ig) G to variant surface antigens (VSAs) seems important for the development of protective immunity against malaria. Unlike VSAs expressed by parasite isolates associated with uncomplicated malaria, VSAs expressed by parasite isolates associated with severe malaria (VSA(SM)) are frequently recognized by IgG. METHODS: We analyzed levels of anti-VSA IgG in 57 individuals in Daraweesh, Sudan, before and after the transmission season. IgG responses to 79 Plasmodium falciparum isolates from children with defined malaria syndromes and exposed to high transmission in a different part of Africa were also analyzed. RESULTS: After the transmission season, individuals with malaria had an increase in IgG recognition to 25.8% (95% confidence interval [CI], 19.9%-31.7%) and a decrease in IgG recognition to 7.6% (95% CI, 4.4%-10.8%) of 79 parasite isolates, and individuals without malaria had an increase in IgG recognition to 8.1% (95% CI, 6.0%-10.2%) and a decrease in IgG recognition to 11.9% (95% CI, 7.0%-16.8%) of 79 parasite isolates. Most newly acquired IgG responses were against parasite isolates expressing VSAs(SM) that are frequently recognized by IgG. CONCLUSIONS: Anti-VSA IgG levels decrease in the absence of infection, and an episode of clinical malaria induces IgG against a range of VSAs, particularly VSAs(SM).     

Autoantibody against dendrite in Plasmodium falciparum infection: a singular auto-immune phenomenon preferentially in cerebral malaria

To investigate auto-reactive antibodies against dendrites of neurons (AAD) previously reported in cerebral malaria (CM) for their functional biological activity, a serological study was conducted in a larger cohort of patients with CM and uncomplicated falciparum malaria (UM). Sera from Thai adults with CM (n = 22) and UM (n = 21) were tested to determine the titers of AAD by indirect fluorescent antibody test and specific antibody responses to Plasmodium falciparum antigens by ELISA. Immunoreactivity against the dendrites of neurons was observed in 100% of sera from the cerebral malaria group as compared to 71% from the non-cerebral malaria group, and the median titer of AAD was higher in CM versus UM, though the difference did not reach significance. In contrast an opposite pattern was seen for anti-P. falciparum antibody titers, which were significantly lower among CM than among UM patients, both for IgG and IgM (p = 0.024 and p = 0.0033, respectively). Our results indicate that this auto-immune phenomenon induced by P. falciparum infection occurs preferentially in cerebral malaria despite lower responses in parasite-specific antibody responses.     

In vitro selection of Plasmodium falciparum 3D7 for expression of variant surface antigens associated with severe malaria in African children

P. falciparum-infected red blood cells (IRBC) can adhere to endothelial host receptors through parasite-encoded, clonally variant surface antigens (VSA). The VSA-mediated IRBC adhesion and the acquired VSA-specific antibody response have both been linked to IRBC organ tropism and disease severity. Parasites isolated from young children with severe malaria (SM) tend to express a limited and conserved set of VSA (VSASM) that are both stronger and more commonly recognized by IgG in the plasma of malaria-exposed individuals than VSA (VSAUM) expressed by parasites causing uncomplicated malaria (UM) in older semi-immune children. Establishment of the genetic mechanism underlying changes in VSA expression in response to in vitro selective pressure is now possible because of the availability of the entire genomic sequence of the P. falciparum clone 3D7. As a first step towards direct molecular identification of VSASM-encoding genes in 3D7, we report here a method of enforcing expression of VSASM-like antigens in this parasite clone by a novel selection method using plasma from semi-immune children with low VSAUM-specific, but high VSASM-specific, IgG reactivity. In addition to the resulting increase in VSA-specific IgG recognition, VSASM-expressing 3D7(3D7-Dodowa1) showed reduced adhesion to CD36. Finally, levels of IgG specific for the VSA expressed by 3D7-Dodowa1 were uniformly higher than those of IgG with specificity for VSA expressed by the unselected 3D7 in plasma samples from geographically and epidemiologically diverse areas of endemic parasite transmission. The described selection method appears a useful tool in the identification of genes encoding VSA involved in severe and life-threatening P. falciparum malaria.     

Plasmodium falciparum malaria in south-west Nigerian children: is the polymorphism of ICAM-1 and E-selectin genes contributing to the clinical severity of malaria?

Plasmodium falciparum malaria remains a major public health hazard in sub-Saharan African children. While the factors that determine the variations in clinical outcome of a malaria have not been completely defined, both host and parasite factors, as well as the complex molecular interactions between them have been implicated. The cyto-adherent properties of the P. falciparum-infected red blood cells are considered as key properties in the pathogenesis of malaria and the polymorphisms of the host adhesion molecules could contribute to the severity of malaria. Clinical information and blood samples were collected from 223 children from Ibadan (south-west Nigeria), median age of 34.5 months, presenting with different clinical manifestations of malaria--clinically asymptomatic parasitism (ACP), acute uncomplicated malaria (UM) and severe malaria (SM)--as defined by WHO criteria. The polymorphisms of genes coding for four human adhesion molecules at six different loci (ICAM-1 exons 2, 4 and 6, E-selectin exon 2, CD36 exon 10, and PECAM exon 3) were studied. DNA samples were prepared for further genotyping of the six exons mentioned above by PCR-RFLPs using the appropriate restriction digests for each loci. The ICAM-1 exon 4 locus was monomorphic. All the other loci were at Hardy-Weinberg equilibrium (HWE).The E-selectin locus had very low heterozygosity (approximately 0.06) in contrast to the other loci under study (0.23-0.44). Once the data was further processed for covariates (age and parasite density) and taking as the reference category the ACP group, results show that in the presence of the G allele at the ICAM-1 exon 6 there is an increased risk (3.6 times) of severe malaria. As far as the T allele in the E-selectin exon is concerned, the number of sampled DNAs with the T allele within both the UM and SM categories is too low for drawing any relevant conclusion at this stage. In conclusion, these results suggest that genetic polymorphisms at host adhesion molecules loci are an important variable in the susceptibility to severe malaria. Further studies of host loci are needed to further delineate which polymorphisms are associated with severe malaria and increase our knowledge of the biology of host-parasite interactions.     

Anti-Plasmodium falciparum antibodies acquired by residents in a holoendemic area of Liberia during development of clinical immunity

Sera from 48 children and adolescents (2-15 years of age), residing in a malaria holoendemic area of Liberia were investigated for specificities and isotypes of anti-P. falciparum antibodies. No clear-cut relationship to the development of clinical immunity was found when the overall antibody activities to total parasite antigens were determined by enzyme-linked immunosorbent assay (ELISA). Although there was a certain rise of IgM, total IgG- and IgG2 antibody activities, this was most pronounced at ages when a clinical but nonsterile immunity is already present. When the sera were investigated by immunoprecipitation of 35S-methionine labeled parasite polypeptides, the total number of parasite antigens precipitated was similar at all ages. Analysis by indirect immunofluorescence (IFA), registering antibodies to intracellular parasite antigens, revealed no age-dependent changes in antibody titers. In contrast, when the sera were assayed by a novel IFA, specific for a restricted number of parasite antigens in the membrane of infected erythrocytes, the frequency of positive sera as well as the anti-P. falciparum titers rose in parallel with the development of clinical immunity. Thus, these antigens appeared to be important inducers of protective immune responses and may be suitable candidates for a vaccine against the asexual blood stages of P. falciparum.     

The use of impregnated curtains does not affect antibody responses against Plasmodium falciparum and complexity of infecting parasite populations in children from Burkina Faso

In Burkina Faso, where malaria is hyper-endemic and transmission intensity is very high, the majority of malaria-related morbidity and mortality occurs in children less than 5 years of age. A control measure such as the use of insecticide-treated curtains (ITC) significantly reduces transmission of malaria infection. Concerns remain whether reduced transmission intensity may lead to a delay in the development of immunity in younger children and even to a partial loss of already acquired immunity. In this study, the levels of P. falciparum-specific IgG subclasses, the number of infecting parasite clones determined by PCR-based genotyping of the msp2 gene and the parasite density were analysed in 154 asymptomatic children (3–6 years) living in 16 villages (8 with and 8 without ITC) in the vicinity of Ouagadougou, the capital of Burkina Faso. In addition, the parasite inhibitory effects of Ig fractions, prepared from selected children, in co-operation with normal human monocytes were studied. Blood samples from asymptomatic ITC-users showed a significant decrease in P. falciparum prevalence as well as in parasite density. However, no significant difference was observed in P. falciparum-specific antibodies or in parasite multiplicity of infection between the two groups. Furthermore, Ig fractions from children of both groups showed similar levels of inhibitory activity against autologous parasite growth both on their own and in co-operation with monocytes.     

Anti-sporozoite antibodies induced by natural infection

Serum samples from 120 individuals living in a malaria-endemic area, 31 patients with Plasmodium falciparum infection, and 58 healthy blood donors were tested for antibodies against P. falciparum and P. vivax sporozoites. Specific antibodies were determined by the circumsporozoite precipitation (CSP) reaction and indirect immunofluorescent (IFA) tests for IgG and IgM antibodies. It was found that a high proportion of adults living in the endemic area had IFA anti-sporozoite antibodies, usually IgG. Children and healthy donors were either negative or had low antibody titers. A positive correlation was found between IgG antibody titers against P. falciparum sporozoites and those against P. vivax sporozoites. CSP reactivity was demonstrated in 5 of 31 sera from patients with falciparum malaria, and was always associated with a high level of IFA antibodies. The anti-sporozoite antibodies were found to be stage- and species-specific.     

Association of intraleukocytic Plasmodium falciparum malaria pigment with disease severity, clinical manifestations, and prognosis in severe malaria

Peripheral parasite density of Plasmodium falciparum is used as an indicator of malaria disease severity, but does not quantify central sequestration, which is important in the pathogenesis of severe disease. Malaria pigment, recognizable within the cytoplasm of phagocytic cells by light microscopy may represent a peripheral marker for parasite biomass. One hundred seventy-two index cases of severe malaria and 172 healthy age-, residence-, and ethnicity-matched controls with uncomplicated malaria in Bandiagara, Mali were analyzed prospectively for presence of malaria pigment. The presence of polymorphonuclear cell (PMN) and monocyte pigment was strongly associated with severe disease compared with uncomplicated malaria. Total PMN pigment burden in children with severe malaria was higher in those with cerebral manifestations and with combined cerebral manifestations and severe anemia (hemoglobin < or = 5 g/dL) but was not associated with hyperparasitemia (> 500,000 asexual forms/mm3). Additionally, pigmented PMNs/mm3 was associated with a fatal outcome in patients with severe malaria. This study validates the presence of malaria pigment in monocytes and neutrophils as a marker for disease severity, and demonstrates that pigmented neutrophils are associated with cerebral malaria and with death in children with severe malaria.     

Anti-schistosome antibody responses in children coinfected with malaria

People residing in schistosome endemic areas are often infected with other parasites. The interaction of the parasites in the host has important implications in the development of acquired immunity to schistosomiasis, and schistosome immuno-epidemiology. An analysis of specific anti-schistosome egg responses in children coinfected with schistosomiasis and malaria shows that malaria positive children produce significantly more anti-schistosome IgE and IgG3 than schistosome infected children who are negative for malaria.     

Placental histopathologic changes associated with subclinical malaria infection and its impact on the fetal environment

Microscopic examination of placental tissue can provide an accurate assessment of malaria infection during pregnancy. In this cross-sectional study of 193 women in Iquitos, Peru, 1.0% and 6.6% had parasites in the peripheral blood as detected by microscopy and polymerase chain reaction, respectively. However, 22% had placental malaria pigment indicating past, subclinical infections. Placental tissues with pigment from 24 cases were matched by gravidity and month of delivery to 24 controls and histopathologically examined. Cases had significantly higher number of monocytes in the intervillous space (44.7 versus 25.5; P = 0.012). Pigmented monocytes in fetal vessels were present in 33.3% of cases. This study demonstrated that subclinical malarial infection occurred frequently in pregnant women and is associated with increased presence of monocytes in the placenta. Pigmented monocytes in fetal vessels suggest parasites can breach the placental barrier and enter the fetal circulation.     

Monocyte activation and cytokine production in Malawian children presenting with P. falciparum malaria

Malaria in malaria‐naïve adults is associated with an inflammatory response characterized by expression of specific activation markers on innate immune cells. Here, we investigate activation and adhesion marker expression, and cytokine production in monocytes from children presenting with cerebral malaria (CM, n = 36), severe malarial anaemia (SMA, n = 42) or uncomplicated malaria (UM, n = 66), and healthy aparasitemic children (n = 52) in Blantyre, Malawi. In all malaria groups, but particularly in the two severe malaria groups, monocyte expression of CD11b, CD11c, CD18, HLA‐DR and CD86, and percentages of TNF‐α‐ and IL‐6‐producing monocytes were lower than in healthy controls, while expression of CD11a, TLR2 and TLR4 was lower in children with severe malaria compared with controls. These levels mostly normalized during convalescence, but percentages of cytokine‐producing monocytes remained suppressed in children with SMA. In all malaria groups, especially the SMA group, a greater proportion of monocytes were loaded with haemozoin than among controls. In a P. falciparum hyperendemic area, monocytes in children with acute symptomatic malaria have reduced expression of adhesion molecules and activation markers and reduced inflammatory cytokine production. This immune suppression could be due to accumulation of haemozoin and/or previous exposure to P. falciparum.     

Serum IgG3 to the Plasmodium falciparum merozoite surface protein 2 is strongly associated with a reduced prospective risk of malaria

The merozoite surface protein 2 (MSP2) of Plasmodium falciparum is recognized by human antibodies elicited during natural infections, and may be a target of protective immunity. In this prospective study, serum IgG antibodies to MSP2 were determined in a cohort of 329 Gambian children immediately before the annual malaria transmission season, and the incidence of clinical malaria in the following 5 months was monitored. Three recombinant MSP2 antigens were used, representing each of the two major allelic serogroups and a conserved region. The prevalence of serum IgG to each antigen correlated positively with age and with the presence of parasitaemia at the time of sampling. These antibodies were associated with a reduced subsequent incidence of clinical malaria during the follow-up. This trend was seen for both IgG1 and IgG3, although the statistical significance was greater for IgG3, the most common subclass against MSP2. After adjusting for potentially confounding effects of age and pre-season parasitaemia, IgG3 reactivities against each of the major serogroups of MSP2 remained significantly associated with a lower prospective risk of clinical malaria. Individuals who had IgG3 reactivity to both of the MSP2 serogroup antigens had an even more significantly reduced risk. Importantly, this effect remained significant after adjusting for a simultaneous strong protective association of antibodies to another antigen (MSP1 block 2) which itself remained highly significant.     

Human antibodies to a Mr 155,000 Plasmodium falciparum antigen efficiently inhibit merozoite invasion.

IgG from a donor clinically immune to Plasmodium falciparum malaria strongly inhibited reinvasion in vitro of human erythrocytes by the parasite. When added to monolayers of glutaraldehyde-fixed and air-dried erythrocytes infected with the parasite, this IgG also displayed a characteristic immunofluorescence restricted to the surface of infected erythrocytes. Elution of the IgG adsorbed to such monolayers gave an antibody fraction that was 40 times more efficient in the reinvasion inhibition assay (50% inhibition titer, less than 1 microgram/ml) than the original IgG preparation. The major antibody in this eluate was directed against a parasite-derived antigen of Mr 155,000 (Pf 155) deposited by the parasite in the erythrocyte membrane in the course of invasion. A detailed study of IgG fractions from 11 donors with acute P. falciparum malaria or clinical immunity revealed the existence of an excellent correlation between their capacities to stain the surface of infected erythrocytes, their titers in reinvasion inhibition, and the presence of antibodies to Pf 155 as detected by immunoblotting. No such correlations were seen when the IgG fractions were analyzed for immunofluorescence of intracellular parasites or for the presence of antibodies to other parasite antigens as detected by immunoprecipitation of [35S]methionine-labeled and NaDodSO4/PAGE-separated parasite extracts. The results suggest that Pf 155 has an important role in the process of erythrocyte infection and that host antibodies to this antigen may efficiently interfere with this process.     

Differential antibody responses to Plasmodium falciparum merozoite proteins in Malawian children with severe malaria

Cerebral malaria (CM) and severe malarial anemia (SMA) are 2 major causes of death in African children infected with Plasmodium falciparum. We investigated levels of naturally acquired antibody to conserved and variable regions of merozoite surface protein (MSP)-1 and MSP-2, apical membrane antigen (AMA)-1, and rhoptry-associated protein 1 in plasma samples from 126 children admitted to the hospital with CM, 59 with SMA, and 84 with uncomplicated malaria (UM) in Malawi. Children with SMA were distinguished by very low levels of immunoglobulin (Ig) G to the conserved C-terminus of MSP-1 and MSP-2 and to full-length AMA-1. Conversely, children with CM had significantly higher levels of IgG to the conserved regions of all antigens examined than did children with UM (for MSP-1 and AMA-1, P< .005; for MSP-2, P< .05) or SMA (for MSP-1 and MSP-2, P<.001; for AMA-1, P< .005). These distinct IgG patterns might reflect differences in age, exposure to P. falciparum, and/or genetic factors affecting immune responses.     

Helicobacter pylori serology at diagnosis and follow-up of biopsy-verified infection in children

Data on the use of Helicobacter pylori serology in children are limited. We studied the serum antibodies of 105 H. pylori-infected children (median age 9.1 y, range 1.5-17.5 y) using an in-house enzyme immunoassay. At diagnosis of the biopsy-verified infection, IgG antibodies to H. pylori were elevated in 98/105 children (93%) but were at a normal level in 7 children, 5 of whom were < 5 y of age. Serum IgA antibodies to H. pylori were elevated in 40/105 children (38%). The levels of IgG and IgA antibody titers to H. pylori correlated with age (p < 0.001 and p < 0.02, respectively). IgG titers were reduced by > or = 50% in 85% (83/98; median follow-up 0.6 y) of children after therapy. In 56 such children eradication was verified by negative histology or urea breath test but I such child showed Helicobacters on histologic examination. Of the 15 children whose IgG titers dropped by < 50%, 7 were considered positive and 4 negative on the basis of histology or urea breath test. In 3 children, IgG titers returned to pretreatment levels 1 y after a 50% drop was seen. Serology is 1 alternative for monitoring H. pylori infection in children, although its sensitivity is lower in very young children. The length of follow-up needed after eradication, however, is unclear.     

Seroepidemiologic evaluation of anti-toxic and anti-colonization factor immunity against infections by LT-producing Escherichia coli in rural Bangladesh

To evaluate serologic immunity against clinical infections by heat-labile enterotoxin-producing Escherichia coli (LT-ETEC) in rural Bangladesh, 124 children and adult women with LT-ETEC diarrhea (cases) were compared with 347 age-matched community controls. In paired acute-convalescent sera from the cases, IgG anti-CFA I and anti-CFA II antibody titers increased eight-to ninefold after infection by LT-ETEC with the homologous CFA, and IgG anti-LT antibody titers increased fourfold for all LT-ETEC infections. Anti-CFA and anti-LT titers peaked in controls aged 12-23 months, the age group with the highest incidence of ETEC infections. However, antibody titers were similar in acute sera from cases and in sera from controls. Although serum IgG anti-CFA and anti-LT antibodies rose in response to LT-ETEC infections and paralleled the age-specific incidence of ETEC in the community, these antibodies were not associated with a lower risk of LT-ETEC diarrhea.     

Elevated levels of nitric oxide and low levels of haptoglobin are associated with severe malarial anaemia in African children

Severe malarial anaemia (SA) is a major complication of malaria and an important cause of child mortality and morbidity. However, the pathogenesis behind SA is poorly understood. Nitric oxide (NO) is known to play a protective role against clinical malaria but is also suggested to have a pathogenic role in cerebral malaria (CM). Erythrophagocytosis by splenic macrophages has been implicated in the pathogenesis of SA. In this study, plasma levels of NO, neopterin, haptoglobin and C-reactive protein (CRP) were measured in paediatric patients with CM, n=77, SA (n=28) and uncomplicated malaria (UM n=53). Haptoglobin levels were significantly lower in SA (median (interquartile range) 25 (17-59) mg/l) than in both CM and UM (40 (24-80) mg/l and 110 (60-160) mg/l, respectively, P<0.001). In contrast, NO levels were higher in SA (38 (28-51) micromol/l) than in CM and UM (21 (15-32) micromol/l and 10.3 (5.6-17) micromol/l, respectively, P<0.001). A significant negative correlation between haptoglobin and NO was seen in the SA group. No such correlation was observed within the UM or CM groups. No significant differences in neopterin levels were observed between any of the three groups, neither was there any correlation between parasitaemias and neopterin levels. The low haptoglobin and high levels of NO in this SA group may contribute to haemolysis. Taken together our results support the hypothesis that immune-mediated erythrocyte destruction is involved in the pathogenesis of malarial anaemia.     

Serum antibody levels to glycosylphosphatidylinositols in specimens derived from matched Malian children with severe or uncomplicated Plasmodium falciparum malaria and healthy controls

Neutralizing antibodies to glycosylphosphatidylinositols (GPIs), which are Plasmodium falciparum surface protein anchor molecules implicated in malaria pathogenesis, are thought to protect against symptomatic malaria. Index cases of severe malaria in Malian children 3 months to 14 years of age were matched by age and residence to uncomplicated malaria and healthy controls. Serum antibodies to GPI (IgM and IgG) were measured at the time of severe malaria and after the malaria transmission season. The mean optical density values for IgM and IgG antibodies were higher in children with severe or uncomplicated malaria compared with healthy controls. Similarly, higher percentages of children with IgM and IgG antibodies to GPI were observed in the severe malaria group compared with matched healthy controls. IgG antibody levels to GPI were highest among children with cerebral malaria and children who died. The IgG antibody levels to GPI peaked during periods of malaria transmission and decreased after malaria transmission ended. A direct correlation between age and parasitemia and IgG antibodies to GPI was observed. In summary, higher levels of IgM and IgG antibodies to GPI in young children were associated with disease severity and were short-lived.