Risk Of Clinical Malaria Research Articles

Activation of transforming growth factor beta by malaria parasite-derived metalloproteinases and a thrombospondin-like molecule.

Much of the pathology of malaria is mediated by inflammatory cytokines (such as interleukin 12, interferon γ, and tumor necrosis factor α), which are part of the immune response that kills the parasite. The antiinflammatory cytokine transforming growth factor (TGF)-β plays a crucial role in preventing the severe pathology of malaria in mice and TGF-β production is associated with reduced risk of clinical malaria in humans. Here we show that serum-free preparations of Plasmodium falciparum, Plasmodium yoelii 17XL, and Plasmodium berghei schizont-infected erythrocytes, but not equivalent preparations of uninfected erythrocytes, are directly able to activate latent TGF-β (LatTGF-β) in vitro. Antibodies to thrombospondin (TSP) and to a P. falciparum TSP-related adhesive protein (PfTRAP), and synthetic peptides from PfTRAP and P. berghei TRAP that represent homologues of TGF-β binding motifs of TSP, all inhibit malaria-mediated TGF-β activation. Importantly, TRAP-deficient P. berghei parasites are less able to activate LatTGF-β than wild-type parasites and their replication is attenuated in vitro. We show that activation of TGF-β by malaria parasites is a two step process involving TSP-like molecules and metalloproteinase activity. Activation of LatTGF-β represents a novel mechanism for direct modulation of the host response by malaria parasites.

The war between the malaria parasite and the immune system: immunity, immunoregulation and immunopathology.

Throughout history malaria has proved to be a significant threat to human health. Between 300 and 500 million clinical cases occur each year worldwide, approximately 2 million of which are fatal, primarily in children. The vast majority of malaria-related deaths are due to infection with Plasmodium falciparum; P. vivax causes severe febrile illness but is rarely fatal. Following repeated exposure to infection, people living in malaria endemic areas gradually acquire mechanisms to limit the inflammatory response to the parasite that causes the acute febrile symptoms (clinical immunity) as well as mechanisms to kill parasites or inhibit parasite replication (antiparasite immunity). Children, who have yet to develop protective immune mechanisms are thus at greater risk of clinical malaria, severe disease and death than adults. However, two epidemiological observations indicate that this is, perhaps, an oversimplified model. Firstly, cerebral malaria - a common manifestation of severe malaria - typically occurs in children who have already acquired a significant degree of antimalarial immunity, as evidenced by lower mean parasite densities and resistance to severe anaemia. One potential explanation is that cerebral malaria is, in part, an immune-mediated disease in which immunological priming occurs during first infection, eventually leading to immunopathology on re-infection. Secondly, among travelers from nonendemic areas, severe malaria is more common - and death rates are higher - in adults than in children. If severe malaria is an immune-mediated disease, what might be priming the immune system of adults from nonendemic areas to cause immunopathology during their first malaria infection, and how do adults from endemic areas avoid severe immunopathology? In this review we consider the role of innate and adaptive immune responses in terms of (i) protection from clinical malaria (ii) their potential role in immunopathology and (iii) the subsequent development of clinical immunity. We conclude by proposing a model of antimalarial immunity which integrates both the immunological and epidemiological data collected to date.

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.

Repeat sequences in block 2 of Plasmodium falciparum merozoite surface protein 1 are targets of antibodies associated with protection from malaria.

Human antibodies to the block 2 region of Plasmodium falciparum merozoite surface protein 1 (MSP1) are associated with a reduced prospective risk of clinical malaria. Block 2 is highly polymorphic, but all known alleles can be grouped into three major types. Two of these types (the K1-like and MAD20-like types) contain type-specific sequences (found in all alleles of a particular type) that flank polymorphic tripeptide repeats. These repeats contain both type-specific and subtype-specific sequences. To evaluate the antibody recognition of these parts of block 2, a new panel of six recombinant proteins was used (fused type-specific flanking sequences and two representative repeat sequences for each of the K1-like and MAD20-like types separately). Extensive testing of these antigens and full-length block 2 antigens showed that human serum immunoglobulin G antibodies induced by infection can recognize (i) type-specific epitopes in the repeats, (ii) subtype-specific epitopes in the repeats, or (iii) type-specific epitopes in flanking sequences. A large prospective study in The Gambia showed that antibodies to the repeats are strongly associated with protection from clinical malaria. The results are important for design of a vaccine to induce protective antibodies, and they address hypotheses about repeat sequences in malaria antigens.

Plasmodium falciparum multiple infections in Mozambique, its relation to other malariological indices and to prospective risk of malaria morbidity.

We describe the frequency of Plasmodium falciparum clones infecting individuals living in a rural area of southern Mozambique and analyse the relationship between multiplicity of infection, age and other malariometric indices, including prospective risk of clinical malaria. The genotyping was based on the use of restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) analysis of P. falciparum merozoite surface protein 2 (msp2). We analysed 826 samples collected during five cross-sectional surveys from residents of Manhiça ranging in age from 4 months to 83 years. We also determined the multiplicity of infection in samples obtained from 6-month-old infants (n = 79) and children <10 years (n = 158) who were then treated and followed prospectively for 1 year or 75 weeks, respectively. Multiplicity of infection did not vary significantly during the first year of life, but increased thereafter, and decreased during adulthood to the levels found in infants. With increasing multiplicity of infection, there was a statistically significant decrease in the risk of submicroscopic infections. There was also a significant correlation between multiplicity of infection and parasite density in infants, children <4 years of age and adults, suggesting that high densities increase the probability of discriminating more clones in complex infections. We found that the relationship between multiple infections and malaria morbidity is age-dependent. In infants, the risk of subsequent episodes of clinical malaria was related to the parasite density but not to baseline multiplicity of infection. In older children, however, the more clones a child carried, the more likely they were to have a clinical malaria episode, and this was true after adjusting for parasite densities. This change in the association between multiplicity and risk of clinical malaria may indicate a shift in the host response to P. falciparum.

Haemoglobin C protects against clinical Plasmodium falciparum malaria.

Haemoglobin C (HbC; beta6Glu --> Lys) is common in malarious areas of West Africa, especially in Burkina Faso. Conclusive evidence exists on the protective role against severe malaria of haemoglobin S (HbS; beta6Glu --> Val) heterozygosity, whereas conflicting results for the HbC trait have been reported and no epidemiological data exist on the possible role of the HbCC genotype. In vitro studies suggested that HbCC erythrocytes fail to support the growth of P. falciparum but HbC homozygotes with high P. falciparum parasitaemias have been observed. Here we show, in a large case-control study performed in Burkina Faso on 4,348 Mossi subjects, that HbC is associated with a 29% reduction in risk of clinical malaria in HbAC heterozygotes (P = 0.0008) and of 93% in HbCC homozygotes (P = 0.0011). These findings, together with the limited pathology of HbAC and HbCC compared to the severely disadvantaged HbSS and HbSC genotypes and the low betaS gene frequency in the geographic epicentre of betaC, support the hypothesis that, in the long term and in the absence of malaria control, HbC would replace HbS in central West Africa.

Environmental risk factors for clinical malaria: a case-control study in the Grau region of Peru

The role of environmental risk factors in clinical malaria has been studied mainly in Africa and Asia, few investigations have been carried out in Latin America. Field observations in northern coastal Peru, where the prevalence of malaria is high during the agricultural season, suggested that the risk of disease varied according to the characteristics of the house and the house environment. Environmental determinants of the risk of clinical malaria were therefore investigated through a case-control study: 323 clinical cases of malaria, recruited through community-based active case-finding, and 969 age-, sex- and village-matched controls were recruited into the study over a period of 12 months ending June 1997. Residual spraying of houses in the previous 6 months, living more than 100 m from a canal, a level of education equal to primary school or above and working in agriculture conferred significant protection from the risk of developing clinical malaria. The presence of spaces between the wall and roof in the subject's bedroom (eaves) and a house aged >4 years statistically significantly increased the risk of disease. Based on these results we discuss possible control measures for malaria in this area of the country.

Fixed, epitope-specific, cytophilic antibody response to the polymorphic block 2 domain of the Plasmodium falciparum merozoite surface antigen MSP-1 in humans living in a malaria-endemic area.

The MSP-1 merozoite surface antigen of the human malaria parasite Plasmodium falciparum is a major target of immune response. The domain called block 2 shows extensive allelic diversity, with more than 50 alleles identified, grouped into three allelic families. Presence of anti-block 2 antibodies has been associated with reduced risk for clinical malaria, but whether or not allele-specific antibodies are implicated remains unclear. To study the fine specificity of the human antibody response, we have used a series of 82 overlapping, N-biotinylated, 15-mer peptides scanning reference alleles and including numerous sequence variants. Peptide antigenicity was validated using sera from mice immunized with recombinant proteins. A cross-sectional survey conducted in a Senegalese village with intense malaria transmission indicated an overall 56 % seroprevalence. The response was specific for individuals and unrelated to the HLA type. Each responder reacted to a few peptides, unrelated to the infecting parasite genotype(s). Seroprevalence of each individual peptide was low, with no identifiable immunodominant epitope. Anti-block 2 antibodies were mostly of the IgG3 isotype, consistent with an involvement in cytophilic antibody-mediated merozoite clearance. The number of responders increased with age, but there was no accumulation of novel specificities with age and hence with exposure to an increasingly large number of alleles. A 15-month longitudinal follow up outlined a remarkably fixed response, with identical reactivity profiles, independent of the past or current parasite types, a pattern reminiscent of clonal imprinting. The implications of the characteristics of the anti-block 2 antibody response in parasite clearance are discussed.

Effect of HIV-1 and increasing immunosuppression on malaria parasitaemia and clinical episodes in adults in rural Uganda: a cohort study

An association between HIV-1 and malaria is expected in theory, but has not been convincingly shown in practice. We studied the effects of HIV-1 infection and advancing immunosuppression on falciparum parasitaemia and clinical malaria. HIV-1-positive and HIV-1-negative adults selected from a population-based cohort in rural Uganda were invited to attend a clinic every 3 months (routine visits) and whenever they were sick (interim visits). At each visit, information was collected on recent fever, body temperature, and malaria parasites. Participants were assigned a clinical stage at each routine visit and had regular CD4-cell measurements. 484 participants made 7220 routine clinic visits between 1990 and 1998. Parasitaemia was more common at visits by HIV-1-positive individuals (328 of 2788 [11.8%] vs 231 of 3688 [6.3%], p<0.0001). At HIV-1-positive visits, lower CD4-cell counts were associated with higher parasite densities, compared with HIV-1-negative visits (p=0.0076). Clinical malaria was significantly more common at HIV-1-positive visits (55 of 2788 [2.0%] vs 26 of 3688 [0.7%], p=0.0003) and the odds of having clinical malaria increased with falling CD4-cell count (p=0.0002) and advancing clinical stage (p=0.0024). Participants made 3377 interim visits. The risk of clinical malaria was significantly higher at visits by HIV-1-positive individuals than HIV-1-negative individuals (4.0% vs 1.9%, p=0.009). The risk of clinical malaria tended to increase with falling CD4-cell counts (p=0.052). HIV-1 infection is associated with an increased frequency of clinical malaria and parasitaemia. This association tends to become more pronounced with advancing immunosuppression, and could have important public-health implications for sub-Saharan Africa.

6. Multiple Plasmodium falciparum infections in Tanzanian infants

Paired blood samples from 99 Tanzanian infants were analysed to examine the infection dynamics of Plasmodium falciparum during the first year of life. Infecting parasites were genotyped by polymerase chain reaction amplification of the polymorphic gene for the merozoite surface protein 2 and subsequent analysis according to the resulting restriction fragment length polymorphism pattern. The same samples served as controls in a parallel case-control study for which an additional blood sample was taken from each child during a fever episode. The relationship of the number of concurrent infections (multiplicity) with age and morbidity was analysed and results were compared to those of a similar study on older children between 2 and 7 years of age, carried out in the same village at the same time. The mean of 2 infecting genotypes per positive blood sample from community surveys was low compared to that in older children, and there was no significant age-dependency of multiplicity within the first year of life. Multiplicity of infection in fever cases was also independent of age. In infants, multiplicity was positively associated with parasite density and risk of clinical malaria, in contrast to the situation in older children (>2 years). The findings help in the understanding of infection dynamics, premunition, and development of semi-immunity in malaria.

8. Effect of iron supplementation and malaria prophylaxis in infants on Plasmodium falciparum genotypes and multiplicity of infection

During a randomized placebo-controlled trial of chemoprophylaxis against Plasmodium falciparum malaria and iron supplementation, in infants living under conditions of intense transmission, all samples of P. falciparum obtained from children aged 5 and 8 months were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis for the msp2 locus. One hundred and six blood samples were analysed for the number of concurrent infections (multiplicity), and the allelic family of each msp2 genotype was determined. Mean multiplicity of infection was, overall, 2·76 infections/child, and it was significantly reduced in infants receiving chemoprophylaxis. This finding might help to explain the rebound effect in morbidity observed after prophylaxis was ended. Iron supplementation did not affect multiplicity of infection. In infants receiving placebo only, or placebo and iron supplementation, a significant positive association was observed between the number of infections and parasite densities (Spearman's ϱ = 0·25, P − 0·047). This association was lost in the group receiving chemoprophylaxis alone, or in combination with iron. This study showed a significant association of FC27-like msp2 alleles with prospective risk of clinical malaria in children (relative risk = 1·487, P = 0·013). Such an association was also found for the present risk of clinical malaria in infants receiving prophylaxis (odds ratio = 3·84, P = 0·026), which might imply that chemoprophylaxis may impair the development of premunition.

IgG3 antibodies to Plasmodium falciparum merozoite surface protein 2 (MSP2): increasing prevalence with age and association with clinical immunity to malaria.

In a cross-sectional survey carried out in west Africa (The Gambia), where Plasmodium falciparum malaria is endemic with seasonal transmission, 178 individuals 1-75 years of age were assessed for their antibody response to the malaria vaccine candidate, merozoite surface protein 2 (MSP2). Total IgG to recombinant antigens representing full-length, repetitive, and group-specific domains of both allelic families of MSP2 was determined by ELISA. The IgG-subclass profile of IgG-positive sera was assessed. Antibody prevalence was age-dependent, reaching a peak during adolescence. In MSP2-seropositive individuals, there was a predominance of cytophilic antibodies (IgG1 and IgG3); IgG1 antibodies were prevalent in children less than 10 years of age, whereas in adolescents and adults MSP2-specific antibodies were predominantly IgG3. In parallel, we conducted a longitudinal study of children (3-8 years of age) from the same community; sera collected before the malaria transmission season were tested for the presence of anti-MSP2 antibodies. The subsequent susceptibility of these children to clinical malaria was monitored and the association between anti-MSP2 antibodies of different IgG subclasses and resistance to clinical malaria was tested. The presence of IgG3 antibodies to MSP2 serogroup A was negatively associated with the risk of clinical malaria whereas IgG1 antibodies to MSP2 serogroup B were associated with an increased risk of clinical infection. Our data suggest that age/exposure-related acquisition of IgG3 antibodies to MSP2 may contribute to the development of clinically protective immunity to malaria.

Immune responses to Plasmodium falciparum antigens during a malaria vaccine trial in Tanzanian children.

Among Tanzanian children living in an area of intense and perennial malaria transmission, prevalence of naturally acquired IgG antibodies that recognize SPf66, NANP, p190 and a 19 kDa fragment of the merozoite surface protein-1 (MSP-1) is high and increases with age. This possibly reflects the high level of natural exposure of the children to P. falciparum. The prevalences of IgG antibodies that recognize the three putative merozoite derived sequences contained in the malaria vaccine SPf66 (83.1, 55.1 and 35.1) is low but also show some age dependence. Three doses of the SPf66 vaccine induce a strong IgG antibody response against both the SPf66 construct, NANP and the three individual peptides. Vaccination with SPf66 did not result in an increase of anti19 kDa fragment antibodies. This reflects the specificity of the humoral immune response induced by the SPf66 construct. Among vaccinated children, antibody titres against SPf66 decreased over time following the third dose. However, 18 months after the third dose, SPf66 recipients still had significantly higher IgG titres and stimulation indices of peripheral blood mononuclear cells (PBMC) than placebo recipients. Within the vaccine group, there is a trend for increasing anti-SPf66 IgG titre to be associated with decreasing risk of clinical malaria but this was not statistically significant. Results also show the difficulties of establishing whether antibody responses are related to protection in field trials in endemic areas.

Mannose binding protein deficiency is not associated with malaria, hepatitis B carriage nor tuberculosis in Africans.

We retrospectively studied MBP genotypes in patients with malaria, tuberculosis (TB), and persistent hepatitis B virus (HBV) carriage, in clinics and hospitals in The Gambia. Children under 10 years with cerebral malaria and/or severe malarial anaemia, were compared with children with symptomatic, mild malaria, and controls of the same age and ethnicity. Adult TB cases with smear-positive pulmonary TB were compared with healthy blood donors from the same ethnic groups. Malaria cases and controls were tested for hepatitis B core antibody (anti-HBc) and surface antigen (HBsAg). TB patients were tested for HIV antibodies. Genotyping used sequence-specific oligonucleotide analysis to identify MBP variant alleles. Overall, 46% (944/2041) of patients and controls were homozygous for the wild-type MBP allele, 45% (922/2041) were carriers of a single variant allele and 8.6% (175/2041) had two variant alleles. Neither homozygotes nor heterozygotes for MBP variants were at increased risk of clinical malaria, persistent HBV carriage or TB. The most common mutation in Africans, the codon 57 variant allele, was weakly associated with resistance to TB (221/794 in TB cases and 276/844 in controls, p = 0.037). MBP deficiency is not a significant risk factor for persistent HBV, severe malaria nor pulmonary TB in West Africa.

Reduced risk of clinical malaria in children infected with multiple clones of Plasmodium falciparum in a highly endemic area: a prospective community study

A prospective community study in a highly malaria endemic area of Papua New Guinea found that infection with multiple Plasmodium falciparum genotypes was an indicator of lowered risk of subsequent clinical attack. The results suggest that concurrent or very recent infections provide protection from superinfecting parasites. The finding of an association between reduced risk of clinical malaria and infection with parasites of merozoite surface protein 1 (MSP-1) type RO33 or MSP-2 type 3D7 further suggests that the concomitant immunity is, at least in part, a consequence of a response to these major merozoite surface proteins.

Mortality and morbidity from malaria after stopping malaria chemoprophylaxis

Gambian children who had received malaria chemoprophylaxis for a variable period of time during their first 5 years of life were followed to determine whether they experienced a rebound in mortality or in morbidity from malaria during the period after chemoprophylaxis was stopped. The risk of dying between the ages of 5 years, when chemoprophylaxis was stopped, and 10 years was no higher among children who had received chemoprophylaxis with Maloprim ® (pyrimethamine plus dapsone) for some period during their first 5 years of life than among children who had received placebo (21 vs. 24 deaths) and the beneficial effect of chemoprophylaxis on mortality observed during the first 5 years of life was sustained. The incidence of clinical attacks of malaria during the year after medication was stopped was significantly higher among children who had previously received Maloprim ® for several years than among children who had previously received placebo. However, at the end of this year, there was no significant difference in spleen rate, parasite rate or packed cell volume between the 2 groups of children. Thus, stopping chemoprophylaxis after a period of several years increased the risk of clinical malaria but did not result in a rebound in mortality in Gambian children. However, the number of deaths recorded was small, so a modest effect on mortality cannot be excluded.

Randomised trial of efficacy of SPf66 vaccine against Plasmodium falciparum malaria in children in southern Tanzania

SummaryEffective, safe antimalarial vaccines have proved elusive. The synthetic polypeptide SPf66 vaccine is based on pre-erythrocytic and asexual blood-stage proteins of Plasmodium falciparum. We report here a randomised double-blind placebo-controlled trial of the efficacy of the SPf66 vaccine against clinical P falciparum malaria in Idete, southern Tanzania, an area of intense perennial malaria transmission.586 children aged 1-5 years received three doses of vaccine (n=274) or placebo (n=312). The incidence and density of parasitaemia were assessed through repeated cross-sectional surveys on subgroups of children. Morbidity was monitored over a 1 year period through passive case detection in all children plus active case detection in a subgroup of 191. An episode of clinical malaria was defined as measured fever (≥37·5°C) and parasite density >20 000/μL.No severe side-effects were seen and the frequency of mild side-effects after the third dose was less than 6%. The vaccine was highly immunogenic and after three doses all vaccine recipients had detectable anti-SPf66 antibodies: the geometric mean index of response was 8·3 in the vaccine group and 0·7 in the placebo group. The incidence of parasitaemia was similar in both groups. 123 children had at least one episode of clinical malaria during the follow-up period after the third dose and annual incidence rates were 0·25 in the vaccine group and 0·35 in the placebo group. Estimated vaccine efficacy was 31% (95% confidence interval 0-52%; p=0·046). After the third dose there were 6 deaths among the study cohort (1 vaccine, 5 placebo). This study confirms that SPf66 is safe, immunogenic and reduces the risk of clinical malaria among children exposed to intense P falciparum transmission.

The use of untreated bednets and malaria infection, morbidity and immunity.

The effect of bednet use was investigated, without undertaking a specific intervention, in four cross-sectional community-based surveys in 10 villages of a highly endemic area of Papua New Guinea. Over half (55%) of the villagers interviewed reported that they had used a bednet on the previous night. In general and after adjustment for age, village and housing characteristics, bednet users, particularly children, had lower parasite prevalences and spleen rates and less enlarged spleens than non-users. However, users were similar to non-users in terms of fever reported for the previous week, axillary temperature, parasite density and haemoglobin level. The prevalence of antibody to the ring erythrocyte surface antigen and the major merozoite surface antigen 2 was lower in users than non-users. The association with malariometric indices and immune responses remained significant when bednet users were compared with non-users in houses without bednets. Thus, untreated bednets do not reduce malaria transmission sufficiently to decrease morbidity. They might paradoxically increase the risk of clinical malaria by lowering the development of humoral immunity.