Anti-phospholipid antibodies in patients with Plasmodium falciparum malaria.

Plasma levels of antibodies against phosphatidylinositol (PI), phosphatidylcholine (PC) and cardiolipin (CL) were measured by enzyme-linked immunosorbent assay (ELISA) in patients from malaria endemic area of Sudan and The Gambia. Some Sudanese adults produced IgM antibodies against all three types of phospholipids (PL) during an acute Plasmodium falciparum infection. The anti-PL antibody titre returned to preinfection levels in most of the donors 30 days after the disease episode. IgG titres against PI, PC and CL were low. In Gambian children with malaria, IgM antibody titres against PI and PC were significantly higher in those with severe malaria than in those with mild malaria. These results show that a proportion of malaria patients produce anti-PL antibodies during infection and that titres of these antibodies are associated with the severity of disease.     

Immunoglobulin G isotype responses to erythrocyte surface-expressed variant antigens of Plasmodium falciparum predict protection from malaria in African children

We assessed immunoglobulin G (IgG) isotype responses to variant surface antigens (VSA) expressed on parasite-infected erythrocytes of a panel of heterologous isolates during and after acute episodes in groups of Gabonese children presenting with either mild or severe Plasmodium falciparum malaria. In the acute and convalescent phases IgG3 and IgG1 anti-VSA antibodies, respectively, predominated. In the absence of infection, the levels of both cytophilic isotypes waned, while those of IgG4 increased, particularly in those admitted with severe malaria. Prospective analyses showed significantly longer delays between malaria attacks associated both (i) with increasing IgG1 responses with specificity for VSA of isolates from children with mild malaria and (ii) with increasing IgG4 responses with specificity for VSA of isolates from children with severe malaria. These findings imply that the predictive value of prospectively measured cross-reactive VSA-specific IgG antibodies with respect to protection against malaria in African children depends both on their isotype and on their fine specificity.     

Strain variation in tumor necrosis factor induction by parasites from children with acute falciparum malaria.

A small proportion of individuals infected with Plasmodium falciparum develop cerebral malaria. Why it affects some infected individuals but not others is poorly understood. Since tumor necrosis factor (TNF) has been implicated strongly in the pathogenesis of cerebral malaria, here we have compared different parasite isolates for their ability to induce TNF production by human mononuclear cells in vitro. Wild isolates were collected from 34 Gambian children with cerebral malaria and 66 children with uncomplicated malaria fever. Cerebral malaria isolates tended to stimulate more TNF production than mild malaria isolates, but there was considerable overlap between the two groups, and the present data provide only limited support for the hypothesis that cerebral malaria is caused by strains of P. falciparum inducing high levels of TNF. However, it is notable that the amounts of TNF induced by different wild isolates from a single locality differed by over 100-fold. The biological significance of this polymorphism deserves further scrutiny in view of the central role that TNF is believed to play in host defense and in the clinical symptomatology of human malaria.     

Increased plasma levels of soluble IL-2R are associated with severe Plasmodium falciparum malaria.

Plasma samples from children with mild and severe Plasmodium falciparum malaria and from children with unrelated diseases were collected to investigate whether the clinical outcome of infection was associated with plasma factors which reflected the activity of different cells of the immune system. Children with severe P. falciparum malaria had significantly higher plasma levels of soluble IL-2R than children with mild malaria. Plasma levels of IL-2R and levels of parasitaemia were significantly correlated. Neither parasitaemia nor plasma levels of tumour necrosis factor-alpha (TNF-alpha), IL-6, lymphotoxin (LT), interferon-gamma (IFN-gamma), IL-4, soluble IL-4R or soluble CD8 differed significantly between the two groups of children with malaria. High plasma levels of soluble CD8 were associated with failure of lymphocytes to produce IFN-gamma in vitro following stimulation with P. falciparum antigen. We conclude that soluble IL-2R is a useful marker of disease severity independently of the association with levels of parasitaemia, and that functional regulation of different lymphocyte subsets occurs during acute malaria episodes.     

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.     

Insecticide-treated bed nets reduce plasma antibody levels and limit the repertoire of antibodies to Plasmodium falciparum variant surface antigens.

The use of insecticide-treated bed nets (ITN) has been documented to reduce malaria morbidity and mortality in areas with endemic malaria, but concerns have been raised that ITN usage could affect the acquisition of malaria immunity. Several lines of evidence have indicated that antibodies against variant surface antigens (VSA) are important in the development of naturally acquired immunity to Plasmodium falciparum malaria and may thus be good indicators of immune status. We have compared the levels of VSA antibodies in plasma from children who have used ITN for 4 years to levels in plasma from children from a nearby village not using ITN. A total of 97 plasma samples were analyzed using 13 different P. falciparum isolates. We found that the children using ITN had significantly lower VSA antibody levels and recognized a smaller proportion of the VSA expressed by the tested parasite isolates than children not using ITN.     

Direct Coombs antiglobulin reactions in Gambian children with Plasmodium falciparum malaria. II. Specificity of erythrocyte-bound IgG.

The cause of direct Coombs (DAT) positivity in West African (Gambian) children has been investigated. Results obtained from a comparison of two groups of primary school children, one from an area hyperendemic for P. falciparum malaria and the other partially protected from malaria, indicate an association between a high incidence of DAT positivity and P. falciparum parasitaemia with raised serum antibody titres to falciparum schizonts. Erythrocytes taken from children with a high IgG DAT titre were subjected to acid elution and the eluates examined for IgG content and antigen specificity. Eluted IgG had specific antibody activity against P. falciparum schizont antigen as demonstrated by means of the indirect fluorescent antibody technique. Cross-reaction was not observed with either P. falciparum gametocytes or P. malariae schizonts. No blood group specificity could be demonstrated in either the red cell eluates or serum taken from children with DAT positive red cells. The results provide convincing evidence which implicates falciparum malaria in the aetiology of Coombs positivity in Gambian children. It is probable that erythrocyte sensitization results from passive attachment of circulating complement-fixing malaria antigen-antibody complexes.     

Antibody response to plasmodium falciparum malaria. Comparisons of immunoglobulin concentrations, antibody titres and the antigenicity of different asexual forms of the parasite

Antibody titres of children and adults living in a malaria endemic region were measured by the indirect fluorescent antibody technique, using fluorescein-conjugated monospecific immunoglobulin antisera. The antigenicity of Plasmodium falciparum trophozoites was compared with that of schizonts obtained by in vitro culture of infected blood. Malarial antibody was detected with antisera to IgG, IgM and, at low levels, to IgA but not with antisera to IgD or IgE. Higher titres were obtained with schizonts as the source of antigen than with trophozoites. Adults in whom IgM antibodies were detected had a mean IgM concentration significantly higher than that for individuals who were IgM antibody negative, and there was a positive correlation between IgG fluorescent antibody titres and total IgG in the children examined. Serial plasma samples from children with acute P. falciparum infections were examined to determine changes in immunoglobulin concentrations, antibody titres and the levels of serum antigens as a result of the infection.     

Immunoglobulin G antibody reactivity to a group A Plasmodium falciparum erythrocyte membrane protein 1 and protection from P. falciparum malaria

Variant surface antigens (VSA) on the surface of Plasmodium falciparum-infected red blood cells play a major role in the pathogenesis of malaria and are key targets for acquired immunity. The best-characterized VSA belong to the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. In areas where P. falciparum is endemic, parasites causing severe malaria and malaria in young children with limited immunity tend to express semiconserved PfEMP1 molecules encoded by group A var genes. Here we investigated antibody responses of Tanzanians who were 0 to 19 years old to PF11_0008, a group A PfEMP1. PF11_0008 has previously been found to be highly transcribed in a nonimmune Dutch volunteer experimentally infected with NF54 parasites. A high proportion of the Tanzanian donors had antibodies against recombinant PF11_0008 domains, and in children who were 4 to 9 years old the presence of antibodies to the PF11_0008 CIDR2beta domain was associated with reduced numbers of malaria episodes. These results indicate that homologues of PF11_0008 are present in P. falciparum field isolates and suggest that PF11_0008 CIDR2beta-reactive antibodies might be involved in protection against malaria episodes.     

Invasion pathways and malaria severity in Kenyan Plasmodium falciparum clinical isolates

The invasion of erythrocytes by Plasmodium falciparum occurs through multiple pathways that can be studied in vitro by examining the invasion of erythrocytes treated with enzymes such as neuraminidase, trypsin, and chymotrypsin. We have studied the invasion pathways used by 31 Kenyan P. falciparum isolates from children with uncomplicated or severe malaria. Six distinct invasion profiles were detected, out of eight possible profiles. The majority of isolates (23 of 31) showed neuraminidase-resistant, trypsin-sensitive invasion, characteristic of the pathway mediated by an unknown parasite ligand and erythrocyte receptor "X." The neuraminidase-sensitive, trypsin-sensitive phenotype consistent with invasion mediated by the binding of parasite ligand erythrocyte binding antigen 175 to glycophorin A, the most common invasion profile in a previous study of Gambian field isolates, was seen in only 3 of 31 Kenyan isolates. No particular invasion profile was associated with severe P. falciparum malaria, and there was no significant difference in the levels of inhibition by the various enzyme treatments between isolates from children with severe malaria and those from children with uncomplicated malaria (P, >0.1 for all enzymes; Mann-Whitney U test). These results do not support the hypothesis that differences in invasion phenotypes play an important role in malaria virulence and indicate that considerable gaps remain in our knowledge of the molecular basis of invasion pathways in natural P. falciparum infections.     

Determinants of variant surface antigen antibody response in severe Plasmodium falciparum malaria in an area of low and unstable malaria transmission

The variant surface antigens (VSA) of infected erythrocytes are important pathogenic markers, a set of variants (VSA(SM)), were assumed to be associated with severe malaria (SM), while SM constitutes clinically diverse forms, such as, severe malarial anemia (SMA) and cerebral malaria (CM). This study was conducted in Eastern Sudan, an area of seasonal and unstable malaria transmission. Parasites and plasma were obtained from patients with different clinical grades of malaria, and flow cytometry was used for analysis of VSA antibody (Ab) response. We found that individuals recognized a broader range of isolates had a higher level of VSA Ab against the recognized isolates (correlation coefficient, 0.727, P<0.001). Unexpectedly, at the time of malaria diagnosis, plasma from patients with CM recognized a significantly larger number of isolates than did the plasma from patients with SMA (P<0.001). Parasites obtained from patients with SMA or from children were better recognized than isolates obtained from patients with uncomplicated malaria or from adults, P<0.001, P=0.021, respectively. Taken together, the above findings suggest that the limitations in the VSA immunoglobulin G repertoire were most probably contributing to the pathogenesis of SMA but not to that of CM.     

Inhibitory immunoglobulin M antibodies to tumor necrosis factor-inducing toxins in patients with malaria.

Cytokines such as tumor necrosis factor alpha (TNF) appear to play an important role in the pathogenesis of malaria. We have previously shown that TNF is produced in response to substances released at schizont rupture, which we have called malaria toxins. In mice these toxins stimulate a T cell-independent antibody response, generating short-lived immunoglobulin M (IgM) antibodies that inhibit the TNF-inducing activity of the toxins. We report here that a similar antibody response is seen in humans. Serum from a European adult infected with Plasmodium falciparum inhibited the induction of TNF by malaria toxins derived from P. falciparum-infected erythrocytes. We found that IgM antibodies were responsible for the inhibitory activity. These inhibitory antibodies could not be detected in convalescent-phase serum collected from the same patient 6 weeks later or in sera from healthy European and African controls. The antibodies appeared to be malaria specific in that they inhibited TNF induction by a variety of P. falciparum isolates but failed to inhibit TNF induction by bacterial lipopolysaccharide or lipoteichoic acid. The inhibitory antibodies bound to liposomes containing phosphatidylinositol but not other phospholipids. Serum from a European adult infected with P. vivax also inhibited the activity of toxins derived from P. falciparum-infected erythrocytes, and this too was mediated by IgM antibodies which were malaria specific and bound to phosphatidylinositol liposomes.     

Differences in human antibody reactivity to Plasmodium falciparum variant surface antigens are dependent on age and malaria transmission intensity in northeastern Tanzania

Plasmodium falciparum variant surface antigens (VSA) are involved in the pathogenesis of malaria. Immunoglobulin G (IgG) with specificity for VSA (anti-VSA IgG) is therefore considered important for acquired immunity. To better understand the nature and dynamics of variant-specific IgG responses at population level, we conducted an immunoepidemiological study in nearby communities in northeastern Tanzania, situated at different altitudes and therefore exposed to different levels of P. falciparum transmission intensity. Samples of plasma and infected red blood cells (IRBC) were collected from 759 individuals aged 0 to 19 years. Plasma levels of IgG with specificity for VSA expressed by a panel of different parasite isolates were measured by flow cytometry, while the ability of plasma to inhibit IRBC adhesion to CD36 was examined in cellular assays. The level and repertoire of the heterologous anti-VSA IgG response developed dramatically in individuals at 1 to 2 years of age in the high-transmission area, reaching a maximum level at around 10 years of age; only a modest further increase was observed among older children and adults. In contrast, at lower levels of malaria transmission, anti-VSA IgG levels were lower and the repertoire was more narrow, and similar age- and transmission-dependent differences were observed with regard to the ability of the plasma samples to inhibit adhesion of IRBC to CD36. These differences indicate a strong and dynamic relationship between malaria exposure and functional characteristics of the variant-specific antibody response, which is likely to be important for protection against malaria.     

The gamma/delta T-cell response to Plasmodium falciparum malaria in a population in which malaria is endemic.

Frequencies and absolute numbers of peripheral gamma/delta T cells have been reported to increase after episodes of Plasmodium falciparum malaria in adults with limited or no previous malaria exposure. In contrast, little is known about the gamma/delta T-cell response to malaria in children from areas where malaria is endemic, who bear the burden of malaria-related morbidity and mortality. We investigated the gamma/delta T-cell response in 19 Ghanaian children from an area of hyperendemic, seasonal malaria transmission. The children presented with cerebral malaria (n = 7), severe malarial anemia (n = 5), or uncomplicated malaria (n = 7) and were monitored from admission until 4 weeks later. We found no evidence of increased frequencies of gamma/delta T cells in any of the patient groups, whereas one adult expatriate studied in Ghana and three adults admitted to the hospital in Copenhagen, Denmark, all with uncomplicated, primary P. falciparum malaria, showed increased gamma/delta T-cell frequencies similar to those previously reported. All patients had lowered absolute numbers of peripheral gamma/delta T cells at admission, changing to increased numbers by days 7 to 14 and then returning to normal levels. The study raises questions regarding age and degree of previous exposure as determinants of malaria-induced gamma/delta T-cell responses.     

Plasmodium falciparum: inhibition/reversal of cytoadherence of Thai isolates to melanoma cells by local immune sera.

The effect of sera on the cytoadherence in vitro of Plasmodium falciparum-infected erythrocytes to melanoma cells was examined. Sera from 19 healthy individuals living in endemic malarious areas in Thailand and 24 patients with P. falciparum malaria were tested against four local P. falciparum isolates. Out of 57 sera examined, 12 (21%) showed significant inhibition (greater than 50%) of cytoadherence for at least one isolate. Anti-malarial IgG antibody titres were determined for all 57 sera and although 11 of the 12 inhibitory sera had relatively high titres, 36 out of 47 sera with similarly high titres showed no significant inhibitory activity. Convalescent sera were no more effective than corresponding acute sera in inhibiting the cytoadherence of erythrocytes infected with any of the four heterologous isolates examined. Sera which significantly inhibited cytoadherence were also capable of reversing cytoadherence, and pooled plasma, from healthy individuals living in malarious areas, was effective in significantly reversing the in vitro cytoadherence of all the five parasite isolates examined. The results confirm the antibody mediated strain-specific nature of the inhibition of cytoadherence and stress the difficulty in selecting immune sera potentially useful for the immunotherapy of cerebral malaria patients in Thailand.     

Prevalence and boosting of antibodies to Plasmodium falciparum glycosylphosphatidylinositols and evaluation of their association with protection from mild and severe clinical malaria

Glycosylphosphatidylinositols (GPIs), the anchor molecules of some membrane proteins of Plasmodium species, have been implicated in the induction of immunopathology during malaria infections. Hence, neutralization of GPIs by antibodies may reduce the severity of clinical attacks of malaria. To test this hypothesis, we have assessed the levels of anti-GPI antibodies in plasma from children and adults living in areas of seasonal malaria transmission in The Gambia. In a prospective study of susceptibility to clinical or asymptomatic infection, the levels of anti-GPI antibodies were measured before and after the transmission season. Samples were also obtained from children recruited into a hospital-based study of severe malaria. We find that in malaria-exposed individuals both the prevalence and the concentration of anti-GPI antibodies increase with age and that antibody levels are significantly higher at the end of the malaria transmission season than at the start of the season. Antibody levels are also higher in children with asymptomatic infections (i.e., those with a degree of clinical immunity) than in children who developed clinical malaria and high parasitemia, although this difference is not statistically significant. Importantly, antibodies appear to be rapidly boosted by clinical malaria infection, but children under the age of two years are seronegative for anti-GPI antibodies, even during an acute infection. While GPIs may be involved in the pathogenesis of human malaria, the data from this study do not provide any strong evidence to support the notion that anti-GPI antibodies confer resistance to mild or severe malarial disease. Further case-control studies, ideally of a prospective nature, are required to elucidate the role of antiglycolipid antibodies in protection from severe malaria.     

Neonatal and maternal immunological responses to conserved epitopes within the DBL-gamma3 chondroitin sulfate A-binding domain of Plasmodium falciparum erythrocyte membrane protein 1

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates the adherence of P. falciparum-infected erythrocytes to placental syncytiotrophoblasts via interactions with chondroitin sulfate A (CSA), a characteristic of pregnancy-associated malaria. Pregnancy-associated malaria predicts increased susceptibility of newborns to malaria, and it is postulated that transplacental passage of parasite antigen induces immune regulatory activity in the neonate. We wished to examine the immune responsiveness to a CSA-binding domain of PfEMP1, the DBL-gamma3 domain, in cord and maternal venous blood obtained from pregnancies with various histories of P. falciparum infection. We assessed in vitro T-cell cytokine and plasma immunoglobulin G (IgG) and IgM responses to four peptides corresponding to highly conserved regions of a DBL-gamma3 domain common to central African parasite isolates. The presence of placental P. falciparum infection at delivery was associated with elevated frequencies of DBL-gamma3 peptide-specific CD3+ interleukin-10-positive T cells in cord blood, while treatment and clearance of infection prior to delivery was associated with elevated frequencies of CD3+ gamma interferon-positive T cells. DBL-gamma3 peptide-specific IgM antibodies were detected in 12 of 60 (20%) cord plasma samples from those born to mothers with P. falciparum infection during pregnancy. Consistent with polyclonal anti-PfEMP1 antibody responses that are associated with protection against pregnancy-associated malaria, the presence of maternal IgG antibodies with specificity for one of the DBL-gamma3 peptides showed a parity-dependent profile. These data demonstrate that peptides corresponding to conserved regions of the DBL-gamma3 domain of PfEMP1 are immunogenic in P. falciparum-infected mothers and their offspring.     

Fresh isolates from children with severe Plasmodium falciparum malaria bind to multiple receptors

The sequestration of Plasmodium falciparum-infected erythrocytes (pRBC) away from the peripheral circulation is a property of all field isolates. Here we have examined the pRBC of 111 fresh clinical isolates from children with malaria for a number of adhesive features in order to study their possible coexpression and association with severity of disease. A large number of adhesion assays were performed studying rosetting, giant rosetting, and binding to CD36, intercellular adhesion molecule 1, platelet endothelial cell adhesion molecule 1, thrombospondin, heparin, blood group A, and immunoglobulins. Suspension assays were performed at the actual parasitemia of the isolate, while all the static adhesion assays were carried out at an equal adjusted parasitemia. The ability to bind to multiple receptors, as well as the ability to form rosettes and giant rosettes, was found to be more frequent among isolates from children with severe versus mild malaria (P = 0.0015). Rosettes and giant rosettes were more frequent for children with severe malaria, and the cell aggregates were larger and tighter, than for those with mild disease (P = 0.0023). Binding of immunoglobulins (97% of isolates) and of heparin (81% of isolates) to infected erythrocytes was common, and binding to heparin and blood group A was associated with severity of disease (P = 0.011 and P = 0.031, respectively). These results support the idea that isolates that bind to multiple receptors are involved in the causation of severe malaria and that several receptor-ligand interactions work synergistically in bringing about severe disease.     

Management of severe falciparum malaria

Plasmodium falciparum is the most common cause of severe and life-threatening malaria. Falciparum malaria causes over one million deaths every year. In Africa, a vast majority of these deaths occur in children under five years of age. The presentation of severe malaria varies with age and geographical distribution. The mortality rate is higher in adults than in children but African children develop neuro-cognitive sequelae following severe malaria more frequently. The management of severe malaria includes prompt administration of appropriate parenteral anti-malarial agents and early recognition and treatment of the complications. In children, the complications include metabolic acidosis (often caused by hypovolaemia), hypoglycaemia, hyperlacticacidaemia, severe anaemia, seizures and raised intracranial pressure. In adults, renal failure and pulmonary oedema are more common causes of death. In contrast, concomitant bacterial infections occur more frequently in children and are associated with mortality in children. Admission to critical or intensive care units may help reduce the mortality, and the frequency and severity of sequelae related to severe malaria.