Federica Verra

Genetics of susceptibility to Plasmodium falciparum: from classical malaria resistance genes towards genome-wide association studies

Plasmodium falciparum represents one of the strongest selective forces on the human genome. This stable and perennial pressure has contributed to the progressive accumulation in the exposed populations of genetic adaptations to malaria. Descriptive genetic epidemiology provides the initial step of a logical procedure of consequential phases spanning from the identification of genes involved in the resistance/susceptibility to diseases, to the determination of the underlying mechanisms and finally to the possible translation of the acquired knowledge in new control tools. In malaria, the rational development of this strategy is traditionally based on complementary interactions of heterogeneous disciplines going from epidemiology to vaccinology passing through genetics, pathogenesis and immunology. New tools including expression profile analysis and genome‐wide association studies are recently available to explore the complex interactions of host–parasite co‐evolution. Particularly, the combination of genome‐wide association studies with large multi‐centre initiatives can overcome the limits of previous results due to local population dynamics. Thus, we anticipate substantial advances in the interpretation and validation of the effects of genetic variation on malaria susceptibility, and thereby on molecular mechanisms of protective immune responses and pathogenesis.

Haemoglobin C protects against clinical Plasmodium falciparum malaria.

Haemoglobin C (HbC; β6Glu → 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; β6Glu → 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 βS gene frequency in the geographic epicentre of βC, support the hypothesis that, in the long term and in the absence of malaria control, HbC would replace HbS in central West Africa.

Breadth and magnitude of antibody responses to multiple Plasmodium falciparum merozoite antigens are associated with protection from clinical malaria.

ABSTRACT Individuals living in areas where malaria is endemic are repeatedly exposed to many different malaria parasite antigens. Studies on naturally acquired antibody-mediated immunity to clinical malaria have largely focused on the presence of responses to individual antigens and their associations with decreased morbidity. We hypothesized that the breadth (number of important targets to which antibodies were made) and magnitude (antibody level measured in a random serum sample) of the antibody response were important predictors of protection from clinical malaria. We analyzed naturally acquired antibodies to five leading Plasmodium falciparum merozoite-stage vaccine candidate antigens, and schizont extract, in Kenyan children monitored for uncomplicated malaria for 6 months (n = 119). Serum antibody levels to apical membrane antigen 1 (AMA1) and merozoite surface protein antigens (MSP-1 block 2, MSP-2, and MSP-3) were inversely related to the probability of developing malaria, but levels to MSP-119 and erythrocyte binding antigen (EBA-175) were not. The risk of malaria was also inversely associated with increasing breadth of antibody specificities, with none of the children who simultaneously had high antibody levels to five or more antigens experiencing a clinical episode (17/119; 15%; P = 0.0006). Particular combinations of antibodies (AMA1, MSP-2, and MSP-3) were more strongly predictive of protection than others. The results were validated in a larger, separate case-control study whose end point was malaria severe enough to warrant hospital admission (n = 387). These findings suggest that under natural exposure, immunity to malaria may result from high titers antibodies to multiple antigenic targets and support the idea of testing combination blood-stage vaccines optimized to induce similar antibody profiles.

Functional deficit of T regulatory cells in Fulani, an ethnic group with low susceptibility to Plasmodium falciparum malaria

Previous interethnic comparative studies on the susceptibility to malaria performed in West Africa showed that Fulani are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups. This lower susceptibility is not associated to classic malaria-resistance genes, and the analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as non-malaria antigens, revealed higher immune reactivity in Fulani. In the present study we compared the expression profile of a panel of genes involved in immune response in peripheral blood mononuclear cells (PBMC) from Fulani and sympatric Mossi from Burkina Faso. An increased expression of T helper 1 (TH1)-related genes (IL-18, IFNγ, and TBX21) and TH2-related genes (IL-4 and GATA3) and a reduced expression of genes distinctive of T regulatory activity (CTLA4 and FOXP3) were observed in Fulani. Microarray analysis on RNA from CD4+CD25+ (T regulatory) cells, performed with a panel of cDNA probes specific for 96 genes involved in immune modulation, indicated obvious differences between the two ethnic groups with 23% of genes, including TGFβ, TGFβRs, CTLA4, and FOXP3, less expressed in Fulani compared with Mossi and European donors not exposed to malaria. As further indications of a low T regulatory cell activity, Fulani showed lower serum levels of TGFβ and higher concentrations of the proinflammatory chemokines CXCL10 and CCL22 compared with Mossi; moreover, the proliferative response of Fulani to malaria antigens was not affected by the depletion of CD25+ regulatory cells whereas that of Mossi was significantly increased. The results suggest that the higher resistance to malaria of the Fulani could derive from a functional deficit of T regulatory cells.

Very large long-term effective population size in the virulent human malaria parasite Plasmodium falciparum.

It has been proposed that the virulent human malaria parasite Plasmodium falciparum underwent a recent severe population bottleneck. In order to test this hypothesis, we estimated the effective population size of this species from the patterns of nucleotide substitution at 23 nuclear protein–coding loci, using a variety of methods based on coalescent theory. Both simple methods and phylogenetically based maximum–likelihood methods yielded the conclusion that the effective population size of this species has been of the order of at least 105 for the past 300 000—400 000 years.

Haemoglobin C and S Role in Acquired Immunity against Plasmodium falciparum Malaria

A recently proposed mechanism of protection for haemoglobin C (HbC; β6Glu→Lys) links an abnormal display of PfEMP1, an antigen involved in malaria pathogenesis, on the surface of HbC infected erythrocytes together with the observation of reduced cytoadhesion of parasitized erythrocytes and impaired rosetting in vitro. We investigated the impact of this hypothesis on the development of acquired immunity against Plasmodium falciparum variant surface antigens (VSA) encoding PfEMP1 in HbC in comparison with HbA and HbS carriers of Burkina Faso. We measured: i) total IgG against a single VSA, A4U, and against a panel of VSA from severe malaria cases in human sera from urban and rural areas of Burkina Faso of different haemoglobin genotypes (CC, AC, AS, SC, SS); ii) total IgG against recombinant proteins of P. falciparum asexual sporozoite, blood stage antigens, and parasite schizont extract; iii) total IgG against tetanus toxoid. Results showed that the reported abnormal cell-surface display of PfEMP1 on HbC infected erythrocytes observed in vitro is not associated to lower anti- PfEMP1 response in vivo. Higher immune response against the VSA panel and malaria antigens were observed in all adaptive genotypes containing at least one allelic variant HbC or HbS in the low transmission urban area whereas no differences were detected in the high transmission rural area. In both contexts the response against tetanus toxoid was not influenced by the β-globin genotype. These findings suggest that both HbC and HbS affect the early development of naturally acquired immunity against malaria. The enhanced immune reactivity in both HbC and HbS carriers supports the hypothesis that the protection against malaria of these adaptive genotypes might be at least partially mediated by acquired immunity against malaria.

Humoral response to the Anopheles gambiae salivary protein gSG6: a serological indicator of exposure to Afrotropical malaria vectors.

Salivary proteins injected by blood feeding arthropods into their hosts evoke a saliva-specific humoral response which can be useful to evaluate exposure to bites of disease vectors. However, saliva of hematophagous arthropods is a complex cocktail of bioactive factors and its use in immunoassays can be misleading because of potential cross-reactivity to other antigens. Toward the development of a serological marker of exposure to Afrotropical malaria vectors we expressed the Anopheles gambiae gSG6, a small anopheline-specific salivary protein, and we measured the anti-gSG6 IgG response in individuals from a malaria hyperendemic area of Burkina Faso, West Africa. The gSG6 protein was immunogenic and anti-gSG6 IgG levels and/or prevalence increased in exposed individuals during the malaria transmission/rainy season. Moreover, this response dropped during the intervening low transmission/dry season, suggesting it is sensitive enough to detect variation in vector density. Members of the Fulani ethnic group showed higher anti-gSG6 IgG response as compared to Mossi, a result consistent with the stronger immune reactivity reported in this group. Remarkably, anti-gSG6 IgG levels among responders were high in children and gradually declined with age. This unusual pattern, opposite to the one observed with Plasmodium antigens, is compatible with a progressive desensitization to mosquito saliva and may be linked to the continued exposure to bites of anopheline mosquitoes. Overall, the humoral anti-gSG6 IgG response appears a reliable serological indicator of exposure to bites of the main African malaria vectors (An. gambiae, Anopheles arabiensis and, possibly, Anopheles funestus) and it may be exploited for malaria epidemiological studies, development of risk maps and evaluation of anti-vector measures. In addition, the gSG6 protein may represent a powerful model system to get a deeper understanding of molecular and cellular mechanisms underlying the immune tolerance and progressive desensitization to insect salivary allergens.

Genetic variation and natural hybridization betweenOrchis laxiflora andOrchis palustris (Orchidaceae)

AbstractGenetic divergence between population samples ofOrchis laxiflora and ofO. palustris from various European locations was studied by electrophoretic analysis of 25 enzyme loci. An average genetic distance of DNei = 1.24 was found between the two taxa, with 12 out of 25 loci showing alternative alleles (diagnostic loci). Genetic heterogeneity was observed within bothO. laxiflora andO. palustris, when northern and southeastern populations were compared, being lower in the former taxon (D = 0.06), than in the latter (D = 0.16). Karyologically, 2n = 36 was found for bothO. laxiflora andO. palustris. O. laxiflora andO. palustris produce hybrids, described asO. ×intermedia. Genotype analysis of several sympatric samples showed the presence of hybrid zones, including F1 hybrids and, in low proportions, recombinant classes, putatively assigned to Fn and backcrosses, as well as a few introgressed individuals of both taxa. These data indicate that hybrids are only partially fertile, with a very limited mixing up of the two parental gene pools; this is also shown by the lack of significant lowering of genetic distances when sympatric and allopatric heterospecific samples are compared. Accordingly,O. laxiflora andO. palustris form a syngameon; nevertheless they can be considered as good taxonomic species, with virtually distinct gene pools, which evolve independently. The genetic variability inO. laxiflora andO. palustris is remarkably low (

Human Genetic Variation Is Associated With Plasmodium falciparum Drug Resistance

\bar H