Exploring Ubiquinone Biosynthesis Inhibition as a Strategy for Improving Atovaquone Efficacy in Malaria.

Atovaquone (AV) acts on the malaria parasite by competing with ubiquinol (UQH2) for its union to the mitochondrial bc1 complex, preventing the ubiquinone-8 and ubiquinone-9 (UQ-8 and UQ-9) redox recycling, which is a necessary step in pyrimidine biosynthesis. This study focused on UQ biosynthesis in Plasmodium falciparum and adopted proof-of-concept research to better elucidate the mechanism of action of AV and improve its efficacy. Initially, UQ biosynthesis was evaluated using several radioactive precursors and chromatographic techniques. This methodology was suitable for studying the biosynthesis of both UQ homologs and its redox state. Additionally, the composition of UQ was investigated in parasites cultivated at different oxygen saturations or in the presence of AV. AV affected the redox states of both UQ-8 and UQ-9 homologs by increasing the levels of the respective reduced forms. Conversely, low-oxygen environments specifically inhibited UQ-9 biosynthesis and increased the antimalarial efficacy of AV. These findings encouraged us to investigate the biological importance and the potential of UQ biosynthesis as a drug target based on its inhibition by 4-nitrobenzoate (4-NB), a 4-hydroxybenzoate (4-HB) analog. 4-NB effectively inhibits UQ biosynthesis and enhances the effects of AV on parasitic growth and respiration rate. Although 4-NB itself exhibits poor antimalarial activity, its 50% inhibitory concentration (IC50) value increased significantly in the presence of a soluble UQ analog, p-aminobenzoic acid (pABA), or 4-HB. These results indicate the potential of AV combined with 4-NB as a novel therapy for malaria and other diseases caused by AV-sensitive pathogens.

Limonene arrests parasite development and inhibits isoprenylation of proteins in Plasmodium falciparum.

Isoprenylation is an essential protein modification in eukaryotic cells. Herein, we report that in Plasmodium falciparum, a number of proteins were labeled upon incubation of intraerythrocytic forms with either [(3)H]farnesyl pyrophosphate or [(3)H]geranylgeranyl pyrophosphate. By thin-layer chromatography, we showed that attached isoprenoids are partially modified to dolichol and other, uncharacterized, residues, confirming active isoprenoid metabolism in this parasite. Incubation of blood-stage P. falciparum treated with the isoprenylation inhibitor limonene significantly decreased the parasites' progression from the ring stage to the trophozoite stage and at 1.22 mM, 50% of the parasites died after the first cycle. Using Ras- and Rap-specific monoclonal antibodies, putative Rap and Ras proteins of P. falciparum were immunoprecipitated. Upon treatment with 0.5 mM limonene, isoprenylation of these proteins was significantly decreased, possibly explaining the observed arrest of parasite development.

Allelic diversity and antibody recognition of Plasmodium falciparum merozoite surface protein 1 during hypoendemic malaria transmission in the Brazilian amazon region.

The polymorphic merozoite surface protein (MSP-1) of Plasmodium falciparum is a major asexual blood-stage malaria vaccine candidate. The impact of allelic diversity on recognition of MSP-1 during the immune response remains to be investigated in areas of hypoendemicity such as the Brazilian Amazon region. In this study, PCR was used to type variable regions, blocks 2, 4, and 10, of the msp-1 gene and to characterize major gene types (unique combinations of allelic types in variable blocks) in P. falciparum isolates collected across the Amazon basin over a period of 12 years. Twelve of the 24 possible gene types were found among 181 isolates, and 68 (38%) of them had more than one gene type. Temporal, but not spatial, variation was found in the distribution of MSP-1 gene types in the Amazon. Interestingly, some gene types occurred more frequently than expected from random assortment of allelic types in different blocks, as previously found in other areas of endemicity. We also compared the antibody recognition of polymorphic (block 2), dimorphic (block 6), and conserved (block 3) regions of MSP-1 in Amazonian malaria patients and clinically immune Africans, using a panel of recombinant peptides. Results were summarized as follows. (i) All blocks were targeted by naturally acquired cytophilic antibodies of the subclasses IgG1 and IgG3, but the balance between IgG1 and IgG3 depended on the subjects' cumulative exposure to malaria. (ii) The balance between IgG1 and IgG3 subclasses and the duration of antibody responses differed in relation to distinct MSP-1 peptides. (iii) Antibody responses to variable blocks 2 and 6 were predominantly type specific, but variant-specific antibodies that target isolate-specific repetitive motifs within block 2 were more frequent in Amazonian patients than in previously studied African populations.

Active isoprenoid pathway in the intra-erythrocytic stages of Plasmodium falciparum: presence of dolichols of 11 and 12 isoprene units.

N-glycosylation of proteins is required for the intra-erythrocytic schizogony of Plasmodium falciparum. In eukaryotic cells, this process involves the transfer of oligosaccharides from a dolichyl pyrophosphate derivative to asparagine residues. We have identified dolichol, dolichyl phosphate and dolichyl pyrophosphate species of 11 and 12 isoprenoid residues by metabolic labelling with [(3)H]farnesyl pyrophosphate, [(3)H]geranylgeranyl pyrophosphate and [(14)C]acetate in the different intra-erythrocytic stages of P. falciparum. This is the first demonstration of short-chain dolichols in the phylum Apicomplexa. The results demonstrate the presence of an active isoprenoid pathway in the intra-erythrocytic stages of P. falciparum. Parasites treated with mevastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, show depressed biosynthesis of dolichol, dolichyl phosphate and isoprenoid pyrophosphate. This effect is observed in all intra-erythrocytic stages of the parasite life cycle, but is most pronounced in the ring stage. N-linked glycosylation of proteins was inhibited in the ring and young-trophozoite stages after mevastatin treatment of parasite cultures. Therefore the isoprenoid pathway may represent a different approach to the development of new anti-malarial drugs.

Antimalarial use of volatile oil from leaves of Virola surinamensis (Rol.) Warb. by Waiãpi Amazon Indians.

The Amazon Indians Waiãpi living in the West of Amapá State of Brazil, treat malaria with an inhalation of vapor obtained from leaves of Viola surinamensis. The essential oil obtained from adult and plantlet leaves was analyzed by GC/MS and 11 monoterpenes, 11 sesquiterpenes and three phenylpropanoids were identified. Plantlet essential oil caused 100% of growth inhibition after 48 h in the development of the young trophozoite to schizont stage and the sesquiterpene nerolidol (100 microg/ml) was identified as one of the active constituents (100% of growth inhibition was obtained). In addition, examination of [U14C]-glucose incorporation showed that activity of nerolidol is related to the inhibition of glycoprotein biosynthesis.

Allelic diversity at the merozoite surface protein-1 locus of Plasmodium falciparum in clinical isolates from the southwestern Brazilian Amazon.

Nucleotide sequences of each variable block in the Plasmodium falciparum merozoite surface protein-1 gene (PfMSP-1) may be grouped into one of two or three possible allelic types, named after the reference isolates MAD20, K1, and RO33. Allelic diversity at this locus basically results from different combinations of allelic types in variable blocks. We used a polymerase chain reaction (PCR)-based strategy to type the variable blocks 2, 4a, 4b, and 10 of the PfMSP-1 gene of P. falciparum isolates from 54 symptomatic malaria patients living in Rondonia, a hypoendemic area in the southwestern Brazilian Amazon. Ten different PfMSP-1 gene types, defined as unique combinations of allelic types in variable blocks, were identified among the 54 isolates. Twenty-one isolates (39%) harbored more than one gene type and two had at least three genetically distinct clones. Hybrid sequences, with a MAD20-type sequence in the 5' segment (4a) and a K1-type sequence in the 3' segment (4b), were quite common in block 4. Direct sequencing of block 4 PCR products revealed a new putative recombination site in four isolates. In contrast with previous studies, the observed distribution of gene types does not deviate significantly from that expected under the null hypothesis of random association between allelic types detected in each variable block. These contradictory data are discussed with reference to the immunoepidemiologic features prevailing in distinct malaria-endemic areas.

The IgG-subclass distribution of naturally acquired antibodies to Plasmodium falciparum, in relation to malaria exposure and severity.

A critical role has been proposed for the switch from non-cytophilic IgG2 to cytophilic antibodies of IgG1 and IgG3 subclasses observed in the humoral immune responses to Plasmodium falciparum of some Africans. These Africans have acquired clinically immunity naturally, after several years of exposure to holo-endemic malaria. In the present study, the possibility that life-long exposure to low levels of malarial endemicity may be associated with changes in the IgG-subclass composition of antibodies to P. falciparum was investigated in a native Amazonian community. The subjects were 138 malaria-exposed but non-infected Karitiana Indians. In a separate investigation, the concentrations of IgG-subclass antibodies in acutely ill patients with severe malaria (N = 22) were compared with those in age- and sex-matched controls who had uncomplicated malaria (N = 44). Plasma concentrations of IgG against a detergent-soluble extract of P. falciparum schizonts were measured by quantitative ELISA, using indirect standardization. Among the Karitiana, the concentrations of anti-parasite antibodies of all subclasses increased with age, and there was no correlation between age and the proportion of such antibodies which was cytophilic. The predominance of cytophilic IgG1 and non-cytophilic IgG2 antibodies in all age-groups of the Karitiana provides an example of an intermediate pattern of immune responses to P. falciparum which contrasts with those previously described in both clinically immune and non-immune populations. Although mean concentrations of cytophilic IgG1 against P. falciparum were significantly higher in the controls than in the patients with severe malaria, there were no significant differences in other IgG subclasses. Lack of exposure to malaria in the past was associated with disease severity (odds ratio = 4.75; 95% confidence interval = 1.31-17.42), and may explain, at least partially, the occurrence of defective, low-IgG1 antibody responses to P. falciparum in those subjects who had severe malaria.

Trypanosoma cruzi tubulin eliminated in the urine of the infected host.

In previous studies we have identified and characterized an 80-kDa Trypanosoma cruzi urinary antigen (UAg) eliminated during acute infection. Polyclonal antibodies raised against this antigen revealed by western blotting and immunoprecipitation analyses showed the existence of another antigenic component of 50-55 kDa in the UAg preparation. The antiserum was also used for screening of a T. cruzi expression library. Sequencing of inserts from selected cDNA clones showed high homology with the 3' end of the T.cruzi beta-tubulin gene sequence encoding for the C-terminus of the protein. The presence of T. cruzi tubulin in the UAg was confirmed by immunoprecipitation of a 50-55-kDa protein from 125I-labeled UAg with monoclonal antibodies (MAbs) to human alpha/beta-tubulin. Interestingly, MAbs recognized radiolabeled T. cruzi tubulin eliminated in the urine of infected mice 24 hr postinoculation of [35S]methionine-labeled viable trypomastigotes. Tubulin found in the urine proved to be of T. cruzi origin because this protein could not be identified in urinary specimens from uninfected animals or mice acutely infected with Leishmania infantum or Toxoplasma gondii. We conclude that tubulin is one of the parasite antigens eliminated in the urine of T. cruzi-infected hosts. This finding may be used to develop a noninvasive procedure for early diagnosis of Chagas' disease.

Humoral immune response to the 72 kDa heat shock protein from Plasmodium falciparum in populations at hypoendemic areas of malaria in western Brazilian Amazon.

The heat-shock protein Pf72/Hsp70-1 from the human malaria parasite Plasmodium falciparum has been suggested as a potential candidate antigen for a multivalent vaccine. We have investigated the prevalence and levels of IgG antibodies to the recombinant protein PfR44, derived from Pf72/Hsp70-1, in individuals from different age groups living in Candeias do Jamari, an Amazonian town characterized by unstable and hypoendemic malaria transmission. Blood were collected from a household-based random sample comprising 241 people and the sera were comparatively tested against recombinant antigen PfR44 and a detergent-soluble extract of P. falciparum (PfAg-T). The prevalence and levels of IgG antibodies to both recombinant and total P. falciparum antigens were positively correlated with cumulative exposure to malaria, as estimated by the age of the individuals and the duration of their stay in the study area. Nevertheless, correlations between antibody responses to Pf72/Hsp70-1 and the acquisition of protective anti-malarial immunity could not be derived from our data.

The isotype composition and avidity of naturally acquired anti-Plasmodium falciparum antibodies: differential patterns in clinically immune Africans and Amazonian patients.

A critical role has been proposed for cytophilic IgG1 and IgG3 subclass antibodies and monocytes and macrophages in antimalarial immunity. Here we compared the 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 IgG, IgM, IgA, and IgG subclass antibodies were assessed. The results are summarized as follows: 1) high-avidity cytophilic antibodies predominated in clinically immune Senegalese subjects; 2) acutely ill Amazonian patients produced high levels of low-avidity cytophilic antibody; 3) such a response was shortlived, since two months later, the concentrations of cytophilic antibodies were significantly lower; 4) however, affinity maturation of IgG antibodies was observed in Amazonian patients two months after the acute malaria attack. A considerable proportion (35-46%) of anti-P. falciparum IgG1 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.

Detection and characterization of antigens in urine of patients with acute, congenital, and chronic Chagas' disease.

Monoclonal antibodies raised against purified Trypanosoma cruzi urinary antigens were used in an enzyme-linked immunosorbent assay (ELISA) capture test for parasite antigens present in urine specimens of Argentinean and Brazilian patients with Chagas' disease. At diagnosis, antigenuria was demonstrated by ELISA in all acutely and congenitally infected infants studied. Moreover, T. cruzi urinary antigens were detected in samples from three of five patients with acute infections and four of five patients with congenital infections following chemotherapy. At least one ELISA-positive urine specimen from each individual was recorded in a longitudinal survey of 12 chronic chagasic patients. The same parasitic antigens (90 to 80 kDa, pI 5.7 to 6.0; 70 to 65 kDa, pI 4.9 to 4.5; 50 to 45 kDa, pI 5.3 to 5.1; and 40 to 35 kDa, pI 4.8 to 4.5) were identified by immunoprecipitation and two-dimensional polyacrylamide gel electrophoresis analysis of urine samples from patients with different forms of chagasic infection. The 90- to 80-kDa urinary protein resembles a trypomastigote-shed antigen. Determination of antigenuria proved valuable for early diagnosis of Chagas' disease and also for diagnosis of chronic cases with conflicting serology.

N-linked glycoproteins are related to schizogony of the intraerythrocytic stage in Plasmodium falciparum.

Although the existence of O-linked oligosaccharide residues in glycoproteins of Plasmodium falciparum has been shown, the existence of N-linked glycoproteins is still a matter of controversy and skepticism. This report demonstrates the unequivocal presence of N-linked glycoproteins in P. falciparum, principally in the ring and young trophozoite stages of the intraerythrocytic cycle. These glycoproteins lose their capacity to bind to concanavalin A-Sepharose after treatment of cultures with tunicamycin under conditions that do not affect protein synthesis. When the glycoproteins were treated with N-Glycanase(R), oligosaccharides were released. It was possible to identify an N-linked glycoprotein of >200 kDa in the ring stage and also N-linked glycoproteins in the range of 200-30 kDa in the trophozoite stage. Treatment of trophozoites with 12 microM tunicamycin inhibited differentiation to the schizont stage. To our knowledge, this is the first report in the literature unequivocally showing N-linked glycoproteins in trophozoites of P. falciparum as well as their importance for the differentiation of the intraerythrocytic stages of this parasite.

The assessment of antibody affinity distribution by thiocyanate elution: a simple dose-response approach.

We describe a simple dose-response approach to assess the affinity distribution of polyclonal antibodies. The proportion of antigen-specific antibodies dissociated by increasing concentrations of the mild chaotropic agent ammonium thiocyanate (NH4SCN) was measured by enzyme immunoassay, and the distribution of tolerances to this agent was presented in a histogram form. Such 'tolerance distribution', which is analogous to that described in classical dose-response bioassays, is proposed as a representation of the actual antibody affinity distribution. To test this approach, we assessed affinity maturation patterns of anti-Plasmodium falciparum IgG antibodies in paired sera obtained from 22 malaria patients during the acute infection and convalescence. We obtained patterns of antibody affinity distributions consistent with those previously described in immunization experiments with the aid of more complex laboratory and computational approaches. Therefore, we suggest the thiocyanate elution technique as an alternative method for rapid assessment of affinity distributions of polyclonal antibodies elicited against complex antigens, readily applicable to large number of serum samples.

High specificity of Trypanosoma cruzi epimastigote ribonucleoprotein as antigen in serodiagnosis of Chagas' disease.

We assessed the performance of an enzyme-linked immunosorbent assay (ELISA) with the Trypanosoma cruzi epimastigote ribosomal fraction (Tulahuen and Y strains) in order to improve the diagnostic specificity of the test. A total of 100 serum samples from patients with chronic Chagas' disease from Brazil and Argentina were studied. Sera from 116 patients, without Chagas' disease, including 10 with active mucocutaneous leishmaniasis and 20 with visceral leishmaniasis, were used as controls. Immunoglobulin G (IgG) antibodies against the ribosomal fraction (ribonucleoproteins [RNPs]) in the ELISA were found in 97% of samples from patients with Chagas' disease. A total of 99% of the sera from patients without the disease were negative, including sera from patients with mucocutaneous and visceral leishmaniases. The distribution of IgG isotypes in randomly chosen serum samples was determined by ELISA; IgG1 and IgG3 were predominant (100% exhibited IgG1 and 85% exhibited IgG3, and 50% also presented the IgG2 isotype. The distribution of the IgG subclasses was confirmed by the Western blot (immunoblot) technique. When total IgG was assayed by Western blot assay, no correlation was found between the pattern of serum reactivity and the clinical features of the patients with Chagas' disease. Therefore, no typical profile of polypeptide recognition could be associated with any clinical form of Chagas' disease (cardiomyopathy or megaviscera). Our results showed that sera from patients with Chagas' disease react with ribosomal antigens and display a typical profile of IgG isotypes (IgG1 plus IgG3). The RNP ELISA seems to have improved specificity compared with those of routine techniques such as the indirect immunofluorescence assay and hemagglutination because it better discriminates between patients with Chagas' disease and patients without the disease. Since sera from patients with leishmaniasis failed to show cross-reactivity with this antigen, the ELISA seems useful for detecting Chagas' disease as well as confirming the nature of sera, when it is doubtful whether the patients has Chagas' disease, by the isotype distribution of IgG.

In situ immunoassay for the assessment of Trypanosoma cruzi interiorization and growth in cultured cells.

Interiorization and multiplication of Trypanosoma cruzi within its host cells are usually assessed by counting parasites in fixed and stained cover slip preparations, a subjective and time-consuming method. Here we describe an immunoenzymatic assay (ELISA) for assessing the number of internalized parasites in infected LLC-MK2 seed on chamber slides (NUNC). ELISA was performed employing a rabbit polyclonal serum against trypomastigote components (MOP) and anti-rabbit IgG conjugated to peroxidase. The bottom of the chamber slide was then detached and processed for quantification of internalized parasites by the conventional method. Data analysis showed a linear relationship between optical densities and number of internalized parasites (r2 = 93.99, p < 0.001). The assay was also efficient to assess inhibition of parasite interiorization induced by the monosaccharide NAc-D-glucosamine.

Antibody response against Plasmodium falciparum exoantigens and somatic antigens: a longitudinal survey in a rural community in Rondônia, western Brazilian Amazon.

Three clinical and sero-epidemiological cross-sectional surveys involving 50 subjects were performed at six-month intervals in Urupá, a rural community characterized by unstable malaria transmission, situated in Rondônia State, Western Brazilian Amazon. Between the surveys, a clinically and parasitologically passive surveillance was established in this community and 48 malaria attacks (28 due to Plasmodium falciparum and 20 due to Plasmodium vivax) were recorded in this cohort of 50 subjects. Serum samples were collected at each survey and tested by enzyme immunoassay (ELISA) for IgG, IgG subclass and IgM antibodies against P. falciparum exoantigens isolated from culture supernatants and detergent-soluble somatic antigens. As expected, both anti-malarial IgG and IgM antibody titres were shown to rise after a malaria outbreak observed during the follow-up period. Nevertheless, in marked contrast with the profile of anti-malarial IgG subclasses described for semi-immune Africans, in this Amazonian community IgG2 antibodies (that are non-cytophilic) against both antigens were shown to predominate over other IgG subclasses. Such overall predominance of IgG2 subclass titres was statistically significant concerning exoantigens, but was of borderline significance in relation to IgG1 antibodies against somatic antigens (p = 0.052). Moreover, highly variable patterns of boosting were observed in antibody responses against both antigens among the patients who suffered P. falciparum malaria attack during the study.

Trypanosoma cruzi: detection of a circulating antigen in urine of chagasic patients sharing common epitopes with an immunodominant repetitive antigen.

A monospecific antiserum raised to a Trypanosoma cruzi recombinant antigen, with tandem repeat of 68 amino acids, was used to screen urine samples of chagasic and nonchagasic patients. The antiserum detected a specific 150-160 kDa antigen in urine of 60% of chronic chagasic patients, but not in urine samples from nonchagasic patients and healthy control individuals. The reactivity to 150-160 kDa urinary antigen could be abolished by adsorption with the recombinant repetitive antigen. These results suggest that 150-160 kDa urinary antigen is a T. cruzi-derived antigen and specific for Chagas' disease.

Detection of antigens in the urine of patients with acute Plasmodium vivax malaria.

Plasmodium antigens were detected by dot-blot assay in the urine of 50 patients infected with Plasmodium vivax. Antigens also were detected in 12/15 patients who no longer had detectable parasitemia, 3 weeks after chemotherapy. Antigenuria was negative 6 weeks after treatment. By Western blotting, four predominant protein antigens were identified in the urine of patients infected with P. vivax: 200, 180, 150, and 110 kDa. The dot-blot technique may prove to be a rapid and inexpensive method for diagnosing malaria in field studies and for clinical evaluation during chemotherapy.