The isotype composition and avidity of naturally acquired anti-plasmodium falciparum antibodies: differential patterns in clinically immune africans and amazonian patients

This work was supported by grants from the UNDP/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases, the Program for Malaria Contrai in the Amazon (PCMAM) of the National Health Foundation (Brazilian Ministry of Health), and the Fundacao para o Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), Brazil.Universidade de São Paulo. Instituto de Ciências Biomédicas. Departamento de Parasitologia. São Paulo, SP, Brazil.Universidade de São Paulo. Instituto de Ciências Biomédicas. Departamento de Parasitologia. São Paulo, SP, Brazil.Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Belém, PA, Brasil.Universidade de São Paulo. Instituto de Ciências Biomédicas. Departamento de Parasitologia. São Paulo, SP, Brazil.A critical role has been proposed for cytophilic IgOl and IgOJ subclass antibodies and monocytes and macrophages in antimalarial immunity. Here we compared lhe isotype composition and avidity of naturally acquired antibodies, as measured by enzyme immunoassay against a detergent-soluble extract of Plasmodium falciparum schizonts, in clinically immune Senegalese adults (n = 33) and semi-immune, adult Amazonian patients (n = 25). Plasma were collected during an acute symptomatic P. falciparum attack and two months later, and in the absence of recrudescence or reinfection. Specific IgO, IgM, IgA, and IgO subclass antibodies were assessed. The results are summarized as follows: 1) high-avidity cytophilic antibodies predorninated in clinically immune Senegalese subjects; 2) acutely ill Amazonian patients produced high levels of low-avidity cytophilic antibody; 3) such a response was shonlived, since two months later, the concentrations of cytophilic antibodies were significantly lower; 4) however, affinity maturation of IgO antibodies was observed in Amazonian patients two months after the acute malaria attack. A considerable proponion (35-46%) of anti-P. falciparum IgOI antibodies produced by African and Amazonian patients was shown to recognize periodate-sensitive carbohydrate epitopes. The potential impact of these findings on the design and evaluation of antimalarial vaccines is discussed

Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum

Abstract Background Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. Methods The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate. Results The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. Conclusions The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria