Dynamics of PfEMP1 Antibody Profile From Birth to 12 Months of Age in Beninese Infants.

BACKGROUND: Plasmodium falciparum-infected erythrocytes bind to specific endothelial cell receptors via members of the PfEMP1 family exported onto the erythrocyte surface. These interactions are mediated by different types of cysteine-rich interdomain region (CIDR) domains found in the N-terminal region of all PfEMP1. CIDRα1 domains bind endothelial protein C receptor (EPCR), CIDRα2-6 domains bind CD36, whereas the receptor specificity of CIDRß/γ/δ domains is unknown. METHODS: In this study, we investigated the level of immunoglobulin (Ig)G targeting the different types of PfEMP1 CIDR during the first year of life. We used plasma collected longitudinally from children of pregnant women who had been followed closely through pregnancy. RESULTS: Antibodies to CIDRα1 domains were more frequent in cord blood compared with antibodies to CIDRα2-6 domains. Higher IgG levels to EPCR-binding CIDRα1 variants positively correlated with the timing of first infections. Antibodies to all PfEMP1 types declined at similar rates to the point of disappearance over the first 6 months of life. At 12 months, children had acquired antibody to all types of CIDR domains, mostly in children with documented P falciparum infections. CONCLUSIONS: These observations agree with the notion that the timing and phenotype of first P falciparum infections in life are influenced by the immune status of the mother.

Genome-wide association study of antibody responses to Plasmodium falciparum candidate vaccine antigens.

We conducted a genome-wide association study (GWAS) of antibody responses directed to three Plasmodium falciparum vaccine candidate antigens (MSP1, MSP2 and GLURP) previously associated with different patterns of protection against malaria infection in Senegalese children. A total of 174 950 single-nucleotide polymorphisms (SNPs) were tested for association with immunoglobulin G1 (IgG1) responses directed to MSP1 and to GLURP and with IgG3 responses to MSP2 FC27 and to MSP2 3D7. We first performed a single-trait analysis with each antibody response and then a multiple-trait analysis in which we analyzed simultaneously the three immune responses associated with the control of clinical malaria episodes. Suggestive associations (P<1 × 10(-4)) were observed for 25 SNPs in MSP1 antibody response analysis or in multiple-trait analysis. According to the strength of their observed associations and their functional role, the following genes are of particular interest: RASGRP3 (2p22.3, P=7.6 × 10(-6)), RIMS1 (6q13, P=2.0 × 10(-5)), MVB12B (9q33.3, P=8.9 × 10(-5)) and GNPTAB (12q23.2, P=7.4 × 10(-5)). Future studies will be required to replicate these findings in other African populations. This work will contribute to the elucidation of the host genetic factors underlying variable immune responses to P. falciparum.

Schistosoma haematobium infection affects Plasmodium falciparum-specific IgG responses associated with protection against malaria.

We have previously shown that antibody responses directed to Plasmodium falciparum merozoite surface protein (MSP)-1, MSP-2 and glutamate-rich protein (GLURP) are associated with anti-malarial protection in residents of the Niakhar area of Senegal. In the same area, urinary schistosomiasis is frequent and we therefore assessed the possible influence of Schistosoma haematobium infection on these protective anti-malarial IgG responses. After adjustment for confounders, we found that the levels of IgG1 directed to MSP1 and GLURP were significantly lower in helminth carriers. The higher circulating levels of interleukin (IL)-10 present in the plasma of co-infected individuals were associated with decreased anti-plasmodial IgG responses, particularly of those directed to MSP-2. Our data thus reveal a modulation of P. falciparum-specific immune responses in the presence of a trematode helminth infection, potentially increasing infected individuals' risk of plasmodial infection or disease.

Low frequency of mutations in the X gene, core promoter and precore region of hepatitis B virus infected Vietnamese.

Numerous mutations in the hepatitis B virus (HBV) genome have been described, but in most cases their role in the pathogenesis of HBV infection is still unclear. Therefore, we analysed specific mutations in HBV-infected Vietnamese patients and assessed their potential relationship with their clinical outcome. A total of 153 HBV-infected Vietnamese patients with well-characterised clinical profiles were enrolled. None of the study participants had a history of alcohol or drug use and none received any antiviral or immunosuppressive therapy before or during the course of this study. The HBx- and core promoter regions were analysed by sequencing. The majority of isolates corresponded to genotype A. The presence of hepatitis B e antigen (HBeAg) was associated with significantly higher viral loads in the chronic HBV-infection group (P = 0.026). Double mutations in the core promoter (1762/1764) were more frequent in those with cancer than in noncancer patients (P < 0.01). Mutations at nucleotide (nt) 1766/1773 were found at low prevalence but with no obvious association to clinical presentation. Cytosine at nt 1858 was predominant but the stop codon mutation in the precore region was not detected. In the study, 4/48 hepatocellular carcinoma (HCC) patients revealed truncated HBx, whilst the serine to alanine mutation (codon 31) of HBx was more prevalent in cancer patients than in asymptomatic HBV carriers (P < 0.01). Thus, the low frequency of mutations indicates the relation of the absence of antiviral pressure in this population. The exclusively found prevalence of certain mutations detected in those with HBV-related carcinoma nevertheless indicates a degree of association with disease progression.

Mosaics of gene variations in the Interleukin-10 gene promoter affect interleukin-10 production depending on the stimulation used.

Interleukin-10 (IL-10), a cytokine involved in many aspects of the immune response shows interindividual variations in their expression. However, genetic variations of the 5'-flanking region of the IL-10 gene (PIL-10) are poorly characterised with respect to different stimuli. New extended haplo- and genotypes are identified present at differing frequencies in three geographically separated populations. Their influence on IL-10 expression have been assessed in vitro after stimulation of leukocytes with lipopolysaccharide (LPS), dibutyryl-cAMP or following immortalisation with Epstein-Barr virus (lymphoblastoid cell line (LCL)). Interindividual differences of IL-10 production were found to be related to single-nucleotide polymorphisms (SNP) haplotype -6752/-6208 in LCLs (P<0.02), and for haplotypes comprising SNPs -6752/-6208/-3538 after LPS stimulation (P<0.03). Carriers of the IL10.G microsatellite with 22, 24 or 26 dinucleotide repeats linked with the -1087G SNP, exhibited the highest levels of IL-10 expression. Contrasting IL-10 secretion patterns were found for IL10.R microsatellite alleles characterised by 15 dinucleotide repeats: after LPS stimulation this allele was associated with high IL-10 production (P<0.007), but with low IL-10 levels in LCLs (P< 0.038). Thus, the effects of mosaics of genetic elements in the PIL-10 on the capacity of leukocytes to produce IL-10 depend on the agent inducing IL-10 expression.

Fcgamma receptor-mediated phagocytosis of Plasmodium falciparum-infected erythrocytes in vitro.

Although convincing evidence exists for the role of immunoglobulin G (IgG) antibodies in immunity to malaria, antibody titres do not usually predict protection. In this study we have assessed the interaction between Plasmodium falciparum-infected erythrocytes (PE), opsonized with immune serum containing different amounts of IgG antibody isotypes, with either THP-1 cells, ex-vivo human monocytes or IIAI.6 transfectant cells expressing Fc(gamma)RIIa-Arg/Arg131 or -His/His131 allotypes. Our results show that PMA-treated THP-1 cells were capable of phagocytosing serum-opsonized PE by Fc(gamma)RI (CD64) and Fc(gamma)RIIa (CD32), acting synergistically. The known Fc(gamma)RIIa polymorphism motivated us to examine its influence on IgG isotype-mediated phagocytosis of opsonized PE with human monocytes and the IIAI.6 transfectant cells expressing either allelic forms. Regardless of the cell type, PE phagocytosis with Fc(gamma)RIIa-His/His131 was highest following opsonization with a predominantly IgG3-containing immune serum pool. In contrast, PE phagocytosis with Fc(gamma)RIIa-Arg/Arg131 tended to be higher with an IgG1-containing pool. These results suggest a genetically determined influence of effector cell phenotype on IgG antibody-pathogen interaction in P. falciparum malaria.

TNFalpha-308A associated with shorter intervals of Plasmodium falciparum reinfections.

A mutation at position -308 of the tumor necrosis factor alpha (TNF-308A) gene promoter has previously been associated with particular manifestations of infectious and non-infectious diseases. In a longitudinal study on malariological parameters in Gabon, TNF promoter variants of 98 children initially presenting with severe Plasmodium falciparum malaria, followed by a total of 504 reinfection events within 52 months, and 100 children initially presenting with mild malaria followed by a total of 342 reinfections were analyzed. Symptomatic P. falciparum reinfections occurred more quickly (median 11 weeks) in carriers of the TNF-308A allele, with severe malaria at enrollment, compared to carriers of other TNF promoter variants (median 16 weeks). The results show that this particular TNF promoter allele increases the risk of reinfection with the malaria parasite P. falciparum.

Plasmodium falciparum: a major role for IgG3 in antibody-dependent monocyte-mediated cellular inhibition of parasite growth in vitro.

In an attempt to identify parasite antigen-specific antibody isotype(s) mediating inhibition of growth in vitro, we tested unfractionated sera and their corresponding purified antibody isotype-containing fractions in in vitro assays with asexual-stage parasites of Plasmodium falciparum in the presence or absence of monocytes. Using affinity purification techniques we fractionated individual and pooled serum samples from semi-immune Gabonese adults, to obtain samples containing either IgG1, 2, 3, and 4, IgG1, 2, and 4, or IgG3 alone, and a non-IgG fraction. Antibodies were quantified spectrophotometrically and the presence of different isotypes in individual fractions was confirmed by protein gel electrophoresis. In the absence of monocytes, we observed inhibition of parasite growth with whole serum and varying levels of either growth enhancement or inhibition with purified Ig-containing fractions. When used in a standardized assay of antibody-dependent cellular inhibition (ADCI) with a monocyte:infected erythrocyte ratio of 1:1, seven of eight serum samples inhibited growth to a mean level of 42%, and the different Ig-containing fractions displayed varying mean levels of inhibition: IgG3, 44%; IgG1--4, 22%; IgG1, 2, and 4, 10%; and non-IgG, - 10%. The results suggest that, among the different isotypes present in the serum of semi-immune individuals, parasite antigen-specific IgG3 in particular may play an important role in controlling parasitemia via an ADCI mechanism involving monocyte- derived mediators.

HLA alleles in relation to specific immunity to liver stage antigen-1 from plasmodium falciparum in Gabon.

Cellular responses to synthetic peptides from the Liver Stage Antigen-1 (LSA-1) from Plasmodium falciparum were determined in 229 Gabonese children. HLA class I and II typing (by PCR-SSP and -RFLP, respectively) revealed that HLA-A*19, -B*17 (and -B*70), -DRB1*05, -DQA1*0102, -DQB1*0602 and -DPB1*0402 were the most frequent types or alleles at each locus. The DQB1*0201 and DQB1*0301 alleles were present at a higher frequency among IL-6 and IFN-gamma responders to the LSA-Rep and LSA-CTL peptides, respectively, and a higher proportion of these responders carried A*19 or B*53. The DRB1*06 type was positively related to the IL-10 production in response to the LSA-CTL peptide, and responders presented mainly A*2. The specificity A*10 was negatively associated with the cellular response to the LSA-J peptide. These results suggest a degree of genetic regulation of specific immune responses by HLA-A, operating at the pre-erythrocytic stage of development of P. falciparum in this Central African population.

Pre-erythrocytic immunity to Plasmodium falciparum: the case for an LSA-1 vaccine.

A vaccine is urgently needed to stem the global resurgence of Plasmodium falciparum malaria. Vaccines targeting the erythrocytic stage are often viewed as an anti-disease strategy. By contrast, infection might be completely averted by a vaccine against the liver stage, a pre-erythrocytic stage during which the parasite multiplies 10000-fold within hepatocytes. Sterilizing immunity can be conferred by inoculating humans with irradiated pre-erythrocytic parasites, and a recombinant pre-erythrocytic vaccine partially protects humans from infection. Liver-stage antigen-1, one of a few proteins known to be expressed by liver-stage parasites, holds particular promise as a vaccine. Studies of naturally exposed populations have consistently related immune responses against this antigen to protection.

HLA-DQB1*0501-restricted Th1 type immune responses to Plasmodium falciparum liver stage antigen 1 protect against malaria anemia and reinfections.

Protective immunity against Plasmodium falciparum requires constant exposure to the pathogen. T cell-mediated immune responses are induced by T cell epitopes of pre-erythrocytic stage antigens of P. falciparum and involve HLA-restricted CD4 and CD8 cells. Cytotoxic T cell responses to a conserved epitope of P. falciparum liver stage antigen (LSA) type 1 are restricted by the HLA class I allele Bw53. The role of HLA class II alleles in mediating cellular responses against P. falciparum LSA-1 has not yet been demonstrated. In a longitudinal study performed for >4 years, associations were found between the HLA class II allele DQB1*0501 and protection from malaria anemia and malarial reinfections in Gabonese children. Children carrying DQB1*0501 had a higher frequency of interferon-gamma responses to LSA-1 T cell epitopes, compared with noncarriers.

[Allelic polymorphism of Plasmodium falciparum MSP-2 gene in blood samples from Gabonese children]. / Polymorphisme allélique du gène MSP-2 de Plasmodium falciparum analysé a partir d'échantillons sanguins d'enfants gabonais.

In this study we have undertaken the molecular analysis of the MSP-2 gene of R falciparum isolates collected from schoolchildren living in the village of Dienga (Gabon). Using conventional microscopy and the polymerase chain reaction, 61% of these children harboured parasites without any symptom of malaria (asymptomatic status). Children with a malaria episode were those with an axillary temperature > or = 37.5 degrees C and a parasitaemia > or =800 parasites/microl of blood. Comparisons of the allelic diversity and distribution of MSP-2 gene were carried out according to the clinical status at the time of sampling. Polymorphism of the MSP-2 gene was large in both clinical groups, both asymptomatic and symptomatic (11 identified alleles). The allele FC27/560bp (base pairs) was found significantly in clinical isolates. Prevalence of the 3D7 family was 68% and 44% in asymptomatic infections and clinical infections, respectively. Multiple P. falciparum genotypes were more predominant in clinical cases (2.96 clones/child with a malaria attack vs 2.01 clones/child with asymptomatic infections). We observed also a reduction of the complexity of infection beyond the age of 10 years. These results are discussed in regard to studies conducted in other areas in Africa.

Plasmodium falciparum liver-stage antigen-1 peptide-specific interferon-gamma responses are not suppressed during uncomplicated malaria in African children.

Liver-stage antigen (LSA)-1 is a candidate vaccine molecule for Plasmodium falciparum malaria, but knowledge of the evolution of naturally acquired immune responses to LSA-1 in African children is lacking. We therefore assessed cellular immune responses to two defined T cell epitopes of LSA-1, during and after uncomplicated P. falciparum malaria in a group of Gabonese children. In terms of their prevalence, interferon (IFN)-gamma responses of peripheral blood mononuclear cells (PBMC) to an LSA-1 N-terminal peptide, T1, were significantly higher when measured during the acute phase compared with convalescence. IFN-gamma responses to the LSA-J (hinge region) peptide showed a similar profile, but at a lower prevalence. Depletion experiments confirmed that CD8+ T cells are a major source of peptide-driven IFN-gamma, but both lymphoproliferation and the production of IL-10 in response to either of the peptides was low in all children at all times. PBMC from 25% of the children failed to produce IFN-gamma in response to either peptide at any time-point. The results suggest that lymphocytes producing IFN-gamma in response to at least one T cell epitope of LSA-1 are most frequent in the peripheral circulation during the acute phase of P. falciparum malaria. Thus, in this case, the generalised suppression of cell-mediated responses which characterises acute malaria does not affect liver-stage antigen-specific IFN-gamma production. These findings imply that measurements of the frequency of parasite antigen-specific cellular immune responses in clinically healthy individuals may represent significant underestimations, which has important implications for the design of field-based vaccine antigen-related studies.

Plasma interleukin-10:Tumor necrosis factor (TNF)-alpha ratio is associated with TNF promoter variants and predicts malarial complications.

In individuals with severe malarial anemia, plasma levels of tumor necrosis factor (TNF)-alpha tend to exceed those of interleukin (IL)-10. In this study, IL-10:TNF plasma level ratios <1 were found to be a risk factor for both cerebral malaria and severe anemia (P=.009), whereas higher IL-10:TNF ratios were observed more frequently in hyperparasitemic individuals. When considering allelic variants of the TNF promoter in children with severe malaria, carriers of the wild type more frequently had an IL-10:TNF ratio >1 (P=.008). In contrast, individuals with a mutation at position -238 of the TNF promoter (TNF(-238A) and TNF(-376A/-238A)) consistently had lower IL-10 than TNF plasma levels (IL-10:TNF ratio <1; P=.003). Our results show that, in children with severe malaria, TNF promoter variants influence the balance of IL-10:TNF in the plasma, which, in turn, affects the outcome in terms of clinical complications.

Low interleukin-12 activity in severe Plasmodium falciparum malaria.

We compared interleukin-12 (IL-12) and other cytokine activities during and after an acute clinical episode in a matched-pair case-control study of young African children who presented with either mild or severe Plasmodium falciparum malaria. The acute-phase, pretreatment plasma IL-12 and alpha interferon (IFN-alpha) levels, as well as the acute-phase mitogen-stimulated whole-blood production capacity of IL-12, were significantly lower in children with severe rather than mild malaria. IL-12 levels, in addition, showed strong inverse correlations both with parasitemia and with the numbers of circulating malaria pigment-containing neutrophils. Acute-phase plasma tumor necrosis factor (TNF) and IL-10 levels were significantly higher in those with severe malaria, and the concentrations of both of these cytokines were positively correlated both with parasitemia and with the numbers of pigment-containing phagocytes in the blood. Children with severe anemia had the highest levels of TNF in plasma. In all the children, the levels in plasma and production capacities of all cytokines normalized when they were healthy and parasite free. The results indicate that severe but not mild P. falciparum malaria in young, nonimmune African children is characterized by down-regulated IL-12 activity, contrasting markedly with the up-regulation of both TNF and IL-10 in the same children. A combination of disturbed phagocyte functions resulting from hemozoin consumption, along with reduced IFN-gamma responses, may contribute to these differential effects.

Human genetic factors related to susceptibility to mild malaria in Gabon.

Several human genetic factors, including red blood cell polymorphisms (ABO blood group, sickle-cell trait, G6PD deficiency) as well as point mutations in the mannose binding protein (MBP) and in the promoter regions of both the TNF-alpha and NOS2 genes, influence the severity of disease due to infection with Plasmodium falciparum. We assessed their impact on mild P. falciparum malaria, as part of a longitudinal investigation of clinical, parasitological and immunological parameters in a cohort of 300 Gabonese schoolchildren. We found the following frequencies: blood group O (0.54), sickle-cell trait (0.23), G6PD deficiency (0.09), MBP gene mutations (0.34), TNF-alpha promoter mutations (at positions -238: 0.17 and -308: 0.22) and NOS2 promoter mutation (0.18). Blood group O or hemoglobin AA were associated with protection against higher parasitemia. Girls with normal G6PD enzyme activity were protected against clinical malaria attacks. In addition, we demonstrated for the first time that the mutation at position -238 of the gene coding for the promoter region of TNF-alpha was positively correlated with the level of the antibody response specific for epitopes of the antigens MSA-2 and RAP-1 of P. falciparum.

Antibody responses to Plasmodium falciparum: evolution according to the severity of a prior clinical episode and association with subsequent reinfection.

We measured sporozoite- and total parasite antigen-specific IgG and IgM antibodies before and after treatment in matched groups of Gabonese children who presented with either mild or severe Plasmodium falciparum malaria. We investigated the influence of various parameters on these antibody responses, including clinical presentation, age, and post-treatment reinfection profiles. IgG but not IgM responses were strongly influenced by both clinical and parasitological status. IgG responses to the repeat region of the circumsporozoite protein, which were low at admission, particularly so in those with severe anemia, increased after treatment but showed no association with either age or reinfection profiles. Total parasite antigen-specific IgG responses were strongly influenced by parasitological status, and also differed significantly when segregated according to clinical status at admission, age, and reinfection histories. Most notably, anti-parasite IgG responses measured when children were parasite-free were higher and a good indicator of recent reinfections in those who presented with mild rather than with severe malaria. The profile of responses in the latter group suggests some immune system dysfunction, which may reflect the induction of tolerance to parasite antigens.

Immune responses against Plasmodium falciparum asexual blood-stage antigens and disease susceptibility in Gabonese and Cameroonian children.

The frequency and level of cellular and humoral responses to seven synthetic peptides from asexual blood stages of Plasmodium falciparum were measured in two cohorts of children living in areas highly endemic for malaria in Gabon and Cameroon. A prospective longitudinal study was conducted for one year in these sites to examine the relationship between specific in vitro immune responses and susceptibility to clinical malaria. Clinical protection was related to high proliferative responses (merozoite surface antigen-1 [MSA-1] and MSA-2 peptides) as well as to elevated antibody levels (schizont extract, MSA-2, and rhoptry-associated protein-1 [RAP-1] peptides) in the village of Dienga, Gabon. Higher response rates of interferon-gamma but lower response rates of tumor necrosis factor-alpha to four and six peptides, respectively, were observed in Dienga than in Pouma that were independent of the older age of the Gabonese children. Age accounted only for the higher prevalence rate in Dienga of the antibody responders to the peptide from Pf155/ring-infected erythrocyte surface antigen (RESA). Our results support the inclusion of epitopes from MSA-1, MSA-2, RAP-1, and Pf155/RESA antigens in a subunit vaccine against malaria, but show that a longitudinal clinical, parasitologic, and immunologic study conducted according to identical criteria in two separate areas may lead to contrasting observations, demonstrating the geographic limitation of the interpretation of such results.

Relationships between malaria prevalence and malaria-related morbidity in school children from two villages in central Africa.

To investigate the relationship between parasite prevalence and malaria-related morbidity, we carried out a comparative study among cohorts of school children from two villages, Dienga, Gabon, and Pouma, Cameroon, both located in malaria-endemic areas. Seven to 17 year-old children attending primary schools were similarly followed-up at each site to evaluate the frequency of malaria attacks. Follow-up involved daily temperature recording (and blood smears in the case of fever) and preparation of blood smears every two weeks. In Pouma, 186 children were followed-up for six months. In Dienga, 228 children were followed-up for nine months. The mean prevalence rate of Plasmodium falciparum infections (as assessed by the blood smears) was twice as high in Pouma compared with Dienga (45.2% versus 26.8%; P < 0.0001), whereas the monthly malaria attack rate (as assessed by the daily surveillance) was twice as high in Dienga compared with Pouma (21.5% versus 41.4%; P = 0.003). The possible implication of several parameters that may differ between the two areas, such as the malaria transmission level, the economical and social status of the inhabitants, the characteristics of infecting parasite strains, and the genetic background of the population, is discussed.

Interferon-gamma responses are associated with resistance to reinfection with Plasmodium falciparum in young African children.

The contribution of T cell-mediated responses was studied with regard to resistance to reinfection in groups of Gabonese children participating in a prospective study of severe and mild malaria due to infection with Plasmodium falciparum. In those admitted with mild malaria, but not in those with severe malaria, production of IFN-gamma by peripheral blood mononuclear cells (PBMC) in response to either liver-stage or merozoite antigen peptides was associated with significantly delayed first reinfections and with significantly lower rates of reinfection. Proliferative or tumor necrosis factor responses to the same peptides showed no such associations. Production of interferon-gamma by PBMC in response to sporozoite and merozoite antigen peptides was observed in a higher proportion of those presenting with mild malaria. Differences in the Th1/Th2 cytokine balance may be linked to the ability to control parasite multiplication in these young children, helping to explain the marked differences observed in both susceptibility to infection as well as in clinical presentation.