A malaria serological map indicating the intersection between parasite antigenic diversity and host antibody repertoires.

A malaria vaccine targeting Plasmodium falciparum remains a strategic goal for malaria control. If a polyvalent vaccine is to be developed, its subunits would probably be chosen based on immunogenicity (concentration of elicited antibodies) and associations of selected antigens with protection. We propose an additional possible selection criterion for the inclusion of subunit antigens; that is, coordination between elicited antibodies. For the quantitative estimation of this coordination, we developed a malaria serological map (MSM). Construction of the MSM was based on three categories of variables: (i) malaria antigens, (ii) total IgG and IgG subclasses, (iii) different sources of plasma. To validate the MSM, in this study, we used four malaria antigens (AMA1, MSP2-3D7, MSP2-FC27 and Pf332-C231) and re-grouped the plasma samples into five pairs of subsets based on age, gender, residence, HbAS and malaria morbidity in 9 years. The plasma total IgG and IgG subclasses to the test antigens were measured, and the whole material was used for the MSM construction. Most of the variables in the MSM were previously tested and their associations with malaria morbidity are known. The coordination of response to each antigens pair in the MSM was quantified as the correlation rate (CR = overall number of significant correlations/total number of correlations × 100 %). Unexpectedly, the results showed that low CRs were mostly associated with variables linked with malaria protection and the antigen eliciting the least CRs was the one associated with protection. The MSM is, thus, of potential value for vaccine design and understanding of malaria natural immunity.

The effect of adjuvants on the immune response induced by a DBL4ɛ-ID4 VAR2CSA based Plasmodium falciparum vaccine against placental malaria.

A vaccine protecting women against placental malaria could be based on the sub-domains of the VAR2CSA antigen, since antibodies against the DBL4ɛ-ID4 subunit of the VAR2CSA protein can inhibit parasite binding to the placental ligand chondroitin sulphate A (CSA). Here we tested the ability of DBL4ɛ-ID4 to induce binding-inhibitory antibodies when formulated with adjuvants approved for human use. We have characterized the immune response of DBL4ɛ-ID4 in combination with Freund's complete and incomplete adjuvant and with three adjuvants currently being used in clinical trials: Montanide(®) ISA 720, Alhydrogel(®) and CAF01. Antibodies induced against DBL4ɛ-ID4 in combination with these adjuvants inhibited parasite binding to CSA from 82% to 99%. Although, different epitope recognition patterns were obtained for the different formulations, all adjuvant combinations induced strong Th1 and Th2 type responses, resulting in IgG with similar binding strength, with to the DBL4ɛ-ID4 antigen. These results demonstrate that the DBL4ɛ-ID4 antigen is highly immunogenic and that binding inhibitory antibodies are induced when formulated with any of the tested adjuvants.

Eleven years of malaria surveillance in a Sudanese village highlights unexpected variation in individual disease susceptibility and outbreak severity.

An analysis is presented of continuous data collected over 11 years based on 1,902,600 person/days of observation on the malaria experience of the people of Daraweesh, a village in eastern Sudan. Malaria transmission is hypo-endemic: the acquisition of clinical immunity with age is not as obvious as in more holo-endemic areas and malaria remained a problem in all age groups throughout the study. However, this population, who are of Fulani origin, showed a distinctly variable level of disease susceptibility. Thirty-two percent of the village never reported malaria symptoms or required malaria treatment while others experienced up to 8 clinical episodes over the 11 years of observation. Malaria incidence was clearly influenced by drought but much less obviously by rainfall. To what extent outbreak patterns are explicable in terms of anopheline factors, and to human immune factors, remains an interesting question for malaria modelling in this, and in other low transmission zones, such as the burgeoning urban areas of modern Africa.

Novel Plasmodium falciparum clones and rising clone multiplicities are associated with the increase in malaria morbidity in Ghanaian children during the transition into the high transmission season.

A survey of Plasmodium falciparum infection and clone multiplicity in Ghanaian children was carried out to study the effect of the onset of the malaria transmission season on disease incidence. Fortnightly blood samples were collected from 40 children living in the rural town of Dodowa, between February and August 1998. P. falciparum parasite densities were calculated and PCR genotyping was carried out using the polymorphic MSP-1 and MSP-2 genes as target loci for the estimation of the number of parasite clones in each sample. The average clone number was estimated using maximum likelihood techniques and the minimum number of clones per patient was analysed for the effects of age, sex, season, minimum number of clones per child, level of parasitaemia and parasite genotype. The statistical analysis indicated that the more clones a child carried, the more likely they were to have a clinical malaria episode. This was true after adjusting for age and season effects and for the measured circulating parasitaemia. The probability of clinical disease also increased if the MSP-1 MAD 20 and the MSP-2 FC 27 alleles were present. This longitudinal analysis thus indicates that the probability of a Ghanaian child having a symptomatic malaria episode is positively associated with both increasing numbers and novel types of P. falciparum clones.

Population genetic analysis of the Plasmodium falciparum erythrocyte binding antigen-175 (eba-175) gene.

The Plasmodium falciparum erythrocyte binding antigen-175 gene (eba-175) has highly divergent allelic segments (Cseg and Fseg) in one part of the gene (region III), but only a small number of single nucleotide polymorphisms (SNPs) in the rest of the sequence. Here, evidence for the possible importance of the Cseg/Fseg dimorphism was sought in a molecular population genetic analysis of the gene. First, allele frequency distributions were determined for the Cseg/Fseg dimorphism and five SNPs in a sample of five populations in Africa. The inter-population variance in frequencies was higher for Cseg/Fseg (F(ST)=0.18) than for the SNPs (F(ST) values from 0.03 to 0.10), but these values were entirely dependent on the inclusion of one particularly divergent population (Sudan). Second, linkage disequilibrium was measured among the intragenic loci. There was the expected trend of declining linkage disequilibrium with increasing molecular distance, but it is notable that the Cseg allele was in absolute linkage disequilibrium with the two flanking SNPs, whereas the Fseg allele was associated with a broader range of SNP haplotypes. Finally, there was no association between the Cseg/Fseg alleles of eba-175 in parasites and the M/N alleles of the glycophorin A erythrocyte receptor in the human subjects.

Differential patterns of human immunoglobulin G subclass responses to distinct regions of a single protein, the merozoite surface protein 1 of Plasmodium falciparum.

Comparisons of immunoglobulin G (IgG) subclass responses to the major polymorphic region and to a conserved region of MSP-1 in three cohorts of African villagers exposed to Plasmodium falciparum revealed that responses to Block 2 are predominantly IgG3 whereas antibodies to MSP-1(19) are mainly IgG1. The striking dominance of IgG3 to Block 2 may explain the short duration of this response and also the requirement for continuous stimulation by malaria infection to maintain clinical immunity.

A principal target of human immunity to malaria identified by molecular population genetic and immunological analyses.

New strategies are required to identify the most important targets of protective immunity in complex eukaryotic pathogens. Natural selection maintains allelic variation in some antigens of the malaria parasite Plasmodium falciparum. Analysis of allele frequency distributions could identify the loci under most intense selection. The merozoite surface protein 1 (Msp1) is the most-abundant surface component on the erythrocyte-invading stage of P. falciparum. Immunization with whole Msp1 has protected monkeys completely against homologous and partially against non-homologous parasite strains. The single-copy msp1 gene, of about 5 kilobases, has highly divergent alleles with stable frequencies in endemic populations. To identify the region of msp1 under strongest selection to maintain alleles within populations, we studied multiple intragenic sequence loci in populations in different regions of Africa and Southeast Asia. On both continents, the locus with the lowest inter-population variance in allele frequencies was block 2, indicating selection in this part of the gene. To test the hypothesis of immune selection, we undertook a large prospective longitudinal cohort study. This demonstrated that serum IgG antibodies against each of the two most frequent allelic types of block 2 of the protein were strongly associated with protection from P. falciparum malaria.

Chronic Plasmodium falciparum infections in an area of low intensity malaria transmission in the Sudan.

Chronic Plasmodium falciparum malaria infections in a Sudanese village, in an area of seasonal and unstable malaria transmission, were monitored and genetically characterized to study the influence of persistent infection on the immunology and epidemiology of low endemicity malaria. During the October-December malaria season of 1996, 51 individuals out of a population of 420 had confirmed and treated P. falciparum malaria in the village of Daraweesh in eastern Sudan. In a cross-sectional survey carried out in December 1996, an additional 6 individuals were found to harbour a microscopically negative but polymerase chain reaction (PCR)-positive P. falciparum infection. On 1 January 1997, a cohort of 43 individuals aged from 9 to 53, recruited from this group of recently malaria-infected individuals agreed to donate fortnightly blood samples for the next 9 months, the first 6 of which constitute the long Sudanese dry season when transmission falls to undetectable levels. Each blood sample was tested for the presence of persistent malaria infection by microscopy and PCR. Parasite-positive samples were genotyped using PCR assays that detect allelic polymorphism at the MSP-1, MSP-2 and GLURP marker gene loci. Of 43 individuals 16 were found to maintain chronic P. falciparum infections which were continuously genetically characterized.

A study of the urban malaria transmission problem in Khartoum.

A study of malaria prevalence and transmission was carried out in Khartoum, the capital of Sudan. The sentinel sites were El manshia, an urban area on the Blue Nile and Ed dekheinat, a lower-income peri-urban area bordering the White Nile. Anopheles arabiensis, the only malaria vector encountered, was present throughout the year although vector density varied seasonally. Plasmodium falciparum was the only species found in El manshia. In Ed dekheinat P. falciparum, Plasmodium ovale and Plasmodium vivax constituted 84.9, 8.2 and 6.9% of the cases, respectively. Plasmodium ovale appears to have recently spread into Khartoum since it has not previously been reported there. We conclude that focal transmission of malaria in the districts bordering both Niles has become established and that the reservoir of human infections has increased in recent years leading to increased risk of malaria epidemics, particularly in the aftermath of seasonal flooding.

Antibodies to variable Plasmodium falciparum-infected erythrocyte surface antigens are associated with protection from novel malaria infections.

In areas of unstable transmission malaria affects all age groups, but the malaria incidence is lower in adults compared to children and teenagers. Under such conditions subclinical Plasmodium falciparum infections are common and some infections are controlled, because blood parasitaemia is maintained at low densities. Here, we test the hypothesis that the presence or absence of antibodies against variant antigens on the surface of P. falciparum-infected erythrocytes protect individuals against some infectious challenges and render them susceptible to others. Plasma collected in Daraweesh, eastern Sudan, before and after the malaria season from individuals who had (susceptible) or did not have malaria (protected) during the season, were tested for reactivity against variant antigens on the surface of nine parasite isolates by flow cytometry. Both protected and susceptible individuals acquired antibodies to variant antigens during the malaria season. The presence of antibody to a Ghanaian isolate before the season was statistically significantly associated with protection against malaria. When considering all nine isolates, the patterns of antibody acquisition differed between susceptible and protected individuals. Together, the results indicate that pre-existing anti-PfEMP1 antibodies can reduce the risk of contracting clinical malaria when challenged by novel parasite clones expressing homologous, but not heterologous variable surface antigens. The results also confirm that antibodies to variant antigens are induced by both clinical and subclinical infections, and that antibodies against several var sero-types are induced during an infection.

The epidemiology of febrile malaria episodes in an area of unstable and seasonal transmission.

This study investigated the epidemiology of uncomplicated falciparum malaria in an area of unstable and seasonal transmission in eastern Sudan. About 90% of malaria morbidity in this region occurs in the months of September to November, and very few malaria cases occur during the intensely arid Sudanese dry season and during years of drought. The malaria situation in the study site, the village of Daraweesh, was analysed during 3 consecutive malaria seasons in 1993-95 during which the 457 inhabitants suffered at total of 436 episodes of falciparum malaria. Using an Andersen-Gill proportional hazard model for recurrent events stratified by family, we have calculated the relative hazard for clinical malaria episodes by age, sex, haemoglobin genotype, blood type and infection in the previous season. The malaria risk was significantly lower in individuals aged 20-88 years than in the 5-19 years age-group. The relative protection due to adulthood varied between seasons (relative risk, RR, 0x34 to 0x67). Serological data were not consistent with the hypothesis that the age difference in incidence was due to differences in exposure. During the 1993 season the malaria incidence in males was lower than in females (RR = 0x75), during the 1994 season the incidences were comparable, whereas males had an increased risk of malaria in 1995 (RR = 1x87). The relative risk in individuals carrying the haemoglobin AS genotype compared to homozygous AA individuals was 0x57.

Analysis of Plasmodium falciparum PfEMP-1/var genes suggests that recombination rearranges constrained sequences.

The var genes of Plasmodium falciparum encode a family of parasite erythrocyte surface antigens, the PfEMP-1 proteins, which function as adhesion ligands for host endothelial and erythrocyte receptors. PfEMP-1 is extremely polymorphic although the extent of this variation in naturally transmitted parasite populations is unclear. We have identified 56 different sequences from the Duffy binding-like (DBL-1) domain of var genes amplified from six different P. falciparum clones isolated from patient infections in a Sudanese village in October-November 1989. These clones have been compared with 25 PfEMP-1 sequences expressed from different var gene loci by the 3D7A clone and 48 PfEMP-1 sequences from different isolates in endemic areas such as Kenya, Brazil, Gambia, Vietnam and Vanuatu to analyse diversity in clonal, local and 'global' P. falciparum populations. Evidence that certain conserved sequences recur in clones from one Sudanese village and in isolates from all over the world suggests that var gene diversity is the result of recombinational reshuffling of a subset of conserved, presumably ancestral sequences. Recurrence of particular var sequence blocks thus leads to 'overlaps' in the PfEMP-1 sequence repertoire of different P. falciparum clones.

Overlapping antigenic repertoires of variant antigens expressed on the surface of erythrocytes infected by Plasmodium falciparum.

Antibodies against variable antigens expressed on the surface of Plasmodium falciparum-infected erythrocytes are believed to be important for protection against malaria. A target for these antibodies is the P. falciparum erythrocyte membrane protein 1, PfEMP1, which is encoded by around 50 var genes and undergoes clonal variation. Using agglutination and mixed agglutination tests and flow cytometry to analyse the recognition of variant antigens on parasitized erythrocytes by plasma antibodies from individuals living in Daraweesh in eastern Sudan, an area of seasonal and unstable malaria transmission, we show that these antibodies recognize different variant antigens expressed by parasites of different genotype. Comparing the levels and acquisition of antibody to variant antigens in pairs of parasite isolates expressing different variant types, there is a correlation between the acquisition of antibodies to some combinations of variant antigens but not to others. These results indicate that (1) a single infection will induce the production of antibodies recognizing several variants of surface-expressed antigens, (2) the repertoire of variable antigens expressed by different parasites is overlapping and the degree of overlap differs between isolates, and (3) the expression of at least some variant antigens is genetically linked.

Sequence diversity of TT virus in geographically dispersed human populations.

TT virus (TTV) is a newly discovered DNA virus originally classified as a member of the Parvoviridae. TTV is transmitted by blood transfusion where it has been reported to be associated with mild post-transfusion hepatitis. TTV can cause persistent infection, and is widely distributed geographically; we recently reported extremely high prevalences of viraemia in individuals living in tropical countries (e.g. 74% in Papua New Guinea, 83% in Gambia; Prescott & Simmonds, New England Journal of Medicine 339, 776, 1998). In the current study we have compared nucleotide sequences from the N22 region of TTV (222 bases) detected in eight widely dispersed human populations. Some variants of TTV, previously classified as genotypes 1a, 1b and 2, were widely distributed throughout the world, while others, such as a novel subtype of type 1 in Papua New Guinea, were confined to a single geographical area. Five of the 122 sequences obtained in this study (from Gambia, Nigeria, Papua New Guinea, Brazil and Ecuador) could not be classified as types 1, 2 or 3, with the variant from Brazil displaying only 46-50% nucleotide (32-35% amino acid) sequence similarity to other variants. This study provides an indication of the extreme sequence diversity of TTV, a characteristic which is untypical of parvoviruses.

Nine-year longitudinal study of antibodies to variant antigens on the surface of Plasmodium falciparum-infected erythrocytes.

PfEMP1 is an antigenically variable molecule which mediates the adhesion of parasitized erythrocytes to a variety of cell types and which is believed to constitute an important target for naturally acquired protective immune responses in malaria. For 9 years we have monitored individuals living in an area of low-intensity, seasonal, and unstable malaria transmission in eastern Sudan, and we have used this database to study the acquisition, specificity, and duration of the antibody response to variant parasitized erythrocyte surface antigens. Both the levels and the spectrum of reactivity of these antibodies varied considerably among individuals, ranging from low levels of antibodies recognizing only few parasitized erythrocyte surface antigens to high levels of broad-specificity antibodies. In general, episodes of clinical malaria were associated with increases in the levels of parasitized erythrocyte surface-specific antibodies that subsided within months of the attack. This response was often, but not always, specific for the antigenic variants expressed by the parasite isolate causing disease. Our study provides evidence that Palciparum falciparum malaria is associated with a short-lived, variant-specific antibody response to PfEMP1-like antigens exposed on the surface of parasitized erythrocytes. Furthermore, our data suggest that the antigenic repertoires of variant antigens expressed by different parasite isolates show considerable overlapping, at least under Sahelian conditions of low-intensity, seasonal, and unstable malaria transmission. Finally, we demonstrate the existence of persistent differences among individuals in the capacity to mount antibody responses to variant surface antigens.

A longitudinal study of human antibody responses to Plasmodium falciparum rhoptry-associated protein 1 in a region of seasonal and unstable malaria transmission.

Rhoptry-associated protein 1 (RAP1) of Plasmodium falciparum is a nonpolymorphic merozoite antigen that is considered a potential candidate for a malaria vaccine against asexual blood stages. In this longitudinal study, recombinant RAP1 (rRAP1) proteins with antigenicity similar to that of P. falciparum-derived RAP1 were used to analyze antibody responses to RAP1 over a period of 4 years (1991 to 1995) of 53 individuals naturally exposed to P. falciparum malaria. In any 1 year during the study, between 23 and 39% of individuals who had malaria developed immunoglobulin G (IgG) antibodies detectable with at least one rRAP1 protein. However, the anti-RAP1 antibody responses were detected only during or shortly after clinical malarial infections. RAP1 antibody levels declined rapidly (within 1 to 2 months) following drug treatment of the infections. No anti-RAP1 antibodies were usually detected a few months after the end of malaria transmission, during the dry season, or by the start of the next malaria season. Thus, RAP1 IgG responses were very short-lived. The short duration of RAP1 antibody response may explain the apparent lack of response in a surprisingly high proportion of individuals after clinical malarial infections. For some individuals who experienced more than one malarial infection, a higher anti-RAP1 antibody response to subsequent infections than to earlier infections was observed. This suggested secondary responses to RAP1 and thus the development of immunological memory for RAP1.

High recombination rate in natural populations of Plasmodium falciparum.

Malaria parasites are sexually reproducing protozoa, although the extent of effective meiotic recombination in natural populations has been debated. If meiotic recombination occurs frequently, compared with point mutation and mitotic rearrangement, linkage disequilibrium between polymorphic sites is expected to decline with increasing distance along a chromosome. The rate of this decline should be proportional to the effective meiotic recombination rate in the population. Multiple polymorphic sites covering a 5-kb region of chromosome 9 (the msp1 gene) have been typed in 547 isolates from six populations in Africa to test for such a decline and estimate its rate in populations of Plasmodium falciparum. The magnitude of two-site linkage disequilibrium declines markedly with increasing molecular map distance between the sites, reaching nonsignificant levels within a map range of 0.3-1.0 kb in five of the populations and over a larger map distance in the population with lowest malaria endemicity. The rate of decline in linkage disequilibrium over molecular map distance is at least as rapid as that observed in most chromosomal regions of other sexually reproducing eukaryotes, such as humans and Drosophila. These results are consistent with the effective recombination rate expected in natural populations of P. falciparum, predicted on the basis of the underlying molecular rate of meiotic crossover and the coefficient of inbreeding caused by self-fertilization events. This is conclusive evidence to reject any hypothesis of clonality or low rate of meiotic recombination in P. falciparum populations. Moreover, the data have major implications for the design and interpretation of population genetic studies of selection on P. falciparum genes.