Changes in protein turnover and resting energy expenditure after treatment of malaria in Gambian children.

To explore the changes in resting energy expenditure (REE) and whole body protein turnover induced by malaria, 23 children aged 6 to 14 y (23.9 +/- 1.0 kg, 1.3 +/- 0.02 m) were studied on three separate days after treatment (d 1, d 2, and 15 d later). REE was assessed by indirect calorimetry (hood), whereas whole body protein turnover was estimated using a single dose of [15N]glycine administered p.o. by measuring the isotopic enrichment of [15N]ammonia in urine over 12 h. Within the first 3.5 h after treatment, the body temperature dropped from 39.8 +/- 0.1 to 37.8 +/- 0.1 degrees C (p < 0.0001), and REE followed the same pattern, decreasing rapidly from 223 +/- 6 to 187 +/- 4 kJ/kg/d (p < 0.0001). Whole body protein synthesis and breakdown were significantly higher during the 1st day (5.65 +/- 0.38 and 6.21 +/- 0.43 g/kg/d, respectively) than at d 15 (2.95 +/- 0.17 and 2.77 +/- 0.2 g/kg/d). It is concluded that Gambian children suffering from an acute episode of malaria have an increased REE averaging 37% of the control value (d 15) and that this was associated with a substantial increase (by a factor of 2) in whole body protein turnover. A rapid normalization of the hypermetabolism and protein hypercatabolism states after treatment was observed.

Localization of HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum.

Localization of human MHC class I-restricted T cell epitopes in the circumsporozoite (CS) protein of the human parasite Plasmodium falciparum is an important objective in the development of antimalarial vaccines. To this purpose, we synthesized a series of overlapping synthetic 20-mer peptides, spanning the entire sequence of the 7G8 CS molecule except for the central repeat B cell domain. The P.f.CS peptides were first tested for their ability to bind to the human MHC class I HLA-A2.1 molecule on T2, a human cell line. Subsequently, the use of a series of shorter peptide analogues allowed us to determine the optimal A2.1 binding sequence present in several of the 20-mers. Binding P.f.CS peptides were further tested for their capacity to activate PBL from HLA-A2.1+ immune donors living in a malaria-endemic area. Specific IFN-gamma production was detected in the supernatant of cultures of PBL from exposed individuals. Cytotoxic T cell lines and clones were derived from the PBL of one responder, and their activity was shown to be HLA-A2.1-restricted and specific for the peptide 334-342 of the CS protein. In addition, double transgenic HLA-A2.1 x human beta 2-microglobulin mice were immunized with peptide 1-10 of the CS protein. T cells derived from immune lymph nodes displayed a peptide-specific HLA-A2.1-restricted cytolytic activity after one in vitro stimulation.

Residual antimalarial concentrations before treatment in patients with malaria from Cambodia: indication of drug pressure.

BACKGROUND: The Thai-Cambodian border has been known as the origin of antimalarial drug resistance for the past 30 years. There is a highly diverse market for antimalarials in this area, and improved knowledge of drug pressure would be useful to target interventions aimed at reducing inappropriate drug use. METHODS: Baseline samples from 125 patients with falciparum malaria recruited for 2 in vivo studies (in Preah Vihear and Pursat provinces) were analyzed for the presence of 14 antimalarials in a single run, by means of a liquid chromatography-tandem mass spectrometry assay. RESULTS: Half of the patients had residual drug concentrations above the lower limit of calibration for at least 1 antimalarial at admission. Among the drugs detected were the currently used first-line drugs mefloquine (25% and 35% of patients) and piperaquine (15% of patients); the first-line drug against vivax malaria, chloroquine (25% and 41% of patients); and the former first-line drug, quinine (5% and 34% patients). CONCLUSIONS: The findings demonstrate that there is high drug pressure and that many people still seek treatment in the private and informal sector, where appropriate treatment is not guaranteed. Promotion of comprehensive behavioral change, communication, community-based mobilization, and advocacy are vital to contain the emergence and spread of parasite resistance against new antimalarials.

Protection against Plasmodium falciparum challenge induced in Aotus monkeys by liver-stage antigen-3-derived long synthetic peptides.

The vaccine potential of Plasmodium falciparum liver stage antigen-3 (LSA3) was investigated in Aotus monkeys using two long synthetic peptides corresponding respectively to an N-terminal non-repeat peptide (NRP) and repeat 2 (R2) region of the LSA3, adjuvanted by ASO2. Both 100-222 (NRP) and 501-596 repeat peptides induced effector B- and T-cell responses in terms of antigen-driven antibodies and/or specific IFN-gamma secretion. Animals challenged with P. falciparum sporozoites were protected following immunization with either the NRP region alone or the NRP combined with the R2 repeat region, as compared with controls receiving the adjuvant alone. These results indicate that the NRP may be sufficient to induce full, sterile protection and confirm the vaccine potential of LSA3 previously demonstrated in chimpanzees and in Aotus.

The N-terminal domain of Plasmodium falciparum circumsporozoite protein represents a target of protective immunity.

The N-terminal domain of the circumsporozoite protein (CSP) has been largely neglected in the search for a malaria vaccine in spite of being a target of inhibitory antibodies and protective T cell responses in mice. Thus, in order to develop this region as a vaccine candidate to be eventually associated with other candidates and, in particular, with the very advanced C-terminal counterpart, synthetic constructs representing N- and C-terminal regions of Plasmodium falciparum and Plasmodium berghei CSP were administered as single or combined formulations in mice. We show that the antisera generated against the combinations inhibit sporozoite invasion of hepatocytes in vitro better than antisera against single peptides. Furthermore, two different P. falciparum CSP N-terminal constructs (PfCS22-110 and PfCS65-110) were recognized by serum samples from people living in malaria-endemic regions. Importantly, recognition of the short N-terminal peptide (PfCS65-110) by sera from children living in a malaria-endemic region was associated with protection from disease. Taken together, these results underline the potential of using such fragments as malaria vaccine candidates.

Longitudinal analyses of immune responses to Plasmodium falciparum derived peptides corresponding to novel blood stage antigens in coastal Kenya

We have recently described 95 predicted alpha-helical coiled-coil peptides derived from putative Plasmodium falciparum erythrocytic stage proteins. Seventy peptides recognized with the highest level of prevalence by sera from three endemic areas were selected for further studies. In this study, we sequentially examined antibody responses to these synthetic peptides in two cohorts of children at risk of clinical malaria in Kilifi district in coastal Kenya, in order to characterize the level of peptide recognition by age, and the role of anti-peptide antibodies in protection from clinical malaria. Antibody levels from 268 children in the first cohort (Chonyi) were assayed against 70 peptides. Thirty-nine peptides were selected for further study in a second cohort (Junju). The rationale for the second cohort was to confirm those peptides identified as protective in the first cohort. The Junju cohort comprised of children aged 1-6 years old (inclusive). Children were actively followed up to identify episodes of febrile malaria in both cohorts. Of the 70 peptides examined, 32 showed significantly (p<0.05) increased antibody recognition in older children and 40 showed significantly increased antibody recognition in parasitaemic children. Ten peptides were associated with a significantly reduced odds ratio (OR) for an episode of clinical malaria in the first cohort of children and two of these peptides (LR146 and AS202.11) were associated with a significantly reduced OR in both cohorts. LR146 is derived from hypothetical protein PFB0145c in PlasmoDB. Previous work has identified this protein as a target of antibodies effective in antibody dependent cellular inhibition (ADCI). The current study substantiates further the potential of protein PFB0145c and also identifies protein PF11_0424 as another likely target of protective antibodies against P. falciparum malaria

Antibody levels against GLURP R2, MSP1 block 2 hybrid and AS202.11 and the risk of malaria in children living in hyperendemic (Burkina Faso) and hypo-endemic (Ghana) areas.

Differences in parasite transmission intensity influence the process of acquisition of host immunity to Plasmodium falciparum malaria and ultimately, the rate of malaria related morbidity and mortality. Potential vaccines being designed to complement current intervention efforts therefore need to be evaluated against different malaria endemicity backgrounds. The associations between antibody responses to the chimeric merozoite surface protein 1 block 2 hybrid (MSP1 hybrid), glutamate-rich protein region 2 (GLURP R2) and the peptide AS202.11, and the risk of malaria were assessed in children living in malaria hyperendemic (Burkina Faso, n = 354) and hypo-endemic (Ghana, n = 209) areas. Using the same reagent lots and standardized protocols for both study sites, immunoglobulin (Ig) M, IgG and IgG sub-class levels to each antigen were measured by ELISA in plasma from the children (aged 6-72 months). Associations between antibody levels and risk of malaria were assessed using Cox regression models adjusting for covariates. There was a significant association between GLURP R2 IgG3 and reduced risk of malaria after adjusting age of children in both the Burkinabe (hazard ratio 0.82; 95 % CI 0.74-0.91, p < 0.0001) and the Ghanaian (HR 0.48; 95 % CI 0.25-0.91, p = 0.02) cohorts. MSP1 hybrid IgM was associated (HR 0.85; 95 % CI 0.73-0.98, p = 0.02) with reduced risk of malaria in Burkina Faso cohort while IgG against AS202.11 in the Ghanaian children was associated with increased risk of malaria (HR 1.29; 95 % CI 1.01-1.65, p = 0.04). These findings support further development of GLURP R2 and MSP1 block 2 hybrid, perhaps as a fusion vaccine antigen targeting malaria blood stage that can be deployed in areas of varying transmission intensity.

CD4(+) CD25(+) Foxp3(+) Regulatory T Cells, Dendritic Cells, and Circulating Cytokines in Uncomplicated Malaria: Do Different Parasite Species Elicit Similar Host Responses?

Clearing blood-stage malaria parasites without inducing major host pathology requires a finely tuned balance between pro- and anti-inflammatory responses. The interplay between regulatory T (Treg) cells and dendritic cells (DCs) is one of the key determinants of this balance. Although experimental models have revealed various patterns of Treg cell expansion, DC maturation, and cytokine production according to the infecting malaria parasite species, no studies have compared all of these parameters in human infections with Plasmodium falciparum and P. vivax in the same setting of endemicity. Here we show that during uncomplicated acute malaria, both species induced a significant expansion of CD4(+) CD25(+) Foxp3(+) Treg cells expressing the key immunomodulatory molecule CTLA-4 and a significant increase in the proportion of DCs that were plasmacytoid (CD123(+)), with a decrease in the myeloid/plasmacytoid DC ratio. These changes were proportional to parasite loads but correlated neither with the intensity of clinical symptoms nor with circulating cytokine levels. One-third of P. vivax-infected patients, but no P. falciparum-infected subjects, showed impaired maturation of circulating DCs, with low surface expression of CD86. Although vivax malaria patients overall had a less inflammatory cytokine response, with a higher interleukin-10 (IL-10)/tumor necrosis factor alpha (TNF-alpha) ratio, this finding did not translate to milder clinical manifestations than those of falciparum malaria patients. We discuss the potential implications of these findings for species-specific pathogenesis and longlasting protective immunity to malaria.

Comparative genomics of the neglected human malaria parasite Plasmodium vivax

The human malaria parasite Plasmodium vivax is responsible for 25 - 40% of the similar to 515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non- human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species.

Urban and suburban malaria in Rondônia (Brazilian Western Amazon) II. Perennial transmissions with high anopheline densities are associated with human environmental changes

Longitudinal entomological surveys were performed in Vila Candelária and adjacent rural locality of Bate Estaca concomitantly with a clinical epidemiologic malaria survey. Vila Candelária is a riverside periurban neighborhood of Porto Velho, capital of the state of Rondônia in the Brazilian Amazon. High anopheline densities were found accompanying the peak of rainfall, as reported in rural areas of the region. Moreover, several minor peaks of anophelines were recorded between the end of the dry season and the beginning of the next rainy season. These secondary peaks were related to permanent anopheline breeding sites resulting from human activities. Malaria transmission is, therefore, observed all over the year. In Vila Candelária, the risk of malaria infection both indoors and outdoors was calculated as being 2 and 10/infecting bites per year per inhabitant respectively. Urban malaria in riverside areas was associated with two factors: (1) high prevalence of asymptomatic carriers in a stable human population and (2) high anopheline densities related to human environmental changes. This association is probably found in other Amazonian urban and suburban communities. The implementation of control measures should include environmental sanitation and better characterization of the role of asymptomatic carriers in malaria transmission.

Multifaceted Role of Heme during Severe Plasmodium falciparum Infections in India

Several immunomodulatory factors are involved in malaria pathogenesis. Among them, heme has been shown to play a role in the pathophysiology of severe malaria in rodents, but its role in human severe malaria remains unclear. Circulating levels of total heme and its main scavenger, hemopexin, along with cytokine/chemokine levels and biological parameters, including hemoglobin and creatinine levels, as well as transaminase activities, were measured in the plasma of 237 Plasmodium falciparum-infected patients living in the state of Odisha, India, where malaria is endemic. All patients were categorized into well-defined groups of mild malaria, cerebral malaria (CM), or severe noncerebral malaria, which included acute renal failure (ARF) and hepatopathy. Our results show a significant increase in total plasma heme levels with malaria severity, especially for CM and malarial ARF. Spearman rank correlation and canonical correlation analyses have shown a correlation between total heme, hemopexin, interleukin-10, tumor necrosis factor alpha, gamma interferon-induced protein 10 (IP-10), and monocyte chemotactic protein 1 (MCP-1) levels. In addition, canonical correlations revealed that heme, along with IP-10, was associated with the CM pathophysiology, whereas both IP-10 and MCP-1 together with heme discriminated ARF. Altogether, our data indicate that heme, in association with cytokines and chemokines, is involved in the pathophysiology of both CM and ARF but through different mechanisms.

Identification of a Highly Antigenic Linear B Cell Epitope within Plasmodium vivax Apical Membrane Antigen 1 (AMA-1)

Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/ disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290-307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies.

Direct effect of Plasmodium vivax recombinant vaccine candidates AMA-1 and MSP-1(19) on the innate immune response

The recombinant apical membrane antigen 1 (AMA-1) and 19-kDa fragment of merozoite surface protein (MSP-1(19)) are the lead candidates for inclusion in a vaccine against blood stages of malaria due to encouraging protective studies in humans and animals. Despite the importance of an efficacious malaria vaccine, vaccine-related research has focused on identifying antigens that result in protective immunity rather than determining the nature of anti-malarial immune effector mechanisms. Moreover, emphasis has been placed on adaptive rather than innate immune responses. In this study, we investigated the effect of Plasmodium vivax vaccine candidates Pv-AMA-1 and Pv-MSP-1(19) on the immune response of malaria-naive donors. Maturation of dendritic cells is altered by Pv-AMA-1 but not Pv-MSP-1(19), as observed by differential expression of cell surface markers. In addition, Pv-AMA-1 induced an increased production of MIP-1 alpha/CCL3 and decreased production of TARC/CCL17 levels in both dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs). Finally, a significant pro-inflammatory response was elicited by Pv-AMA-1-stimulated PBMCs. These results suggest that the recombinant vaccine candidate Pv-AMA-1 may play a direct role on innate immune response and might be involved in parasite destruction. (C) 2007 Elsevier Ltd. All rights reserved.

Pulmonary toxicity with mefloquine.

This report presents a case of acute lung injury developing within hours after administration of mefloquine for a low-level Plasmodium falciparum malaria, which was persistent despite halofantrine therapy. Extensive microbiological investigation remained negative and video-assisted thoracoscopic lung biopsy demonstrated diffuse alveolar damage. The evolution was favourable without treatment. This is the second report of acute lung injury and diffuse alveolar damage caused by mefloquine. Glucose-6-phosphate dehydrogenase deficiency was present in the former case and was thought to contribute to the lung injury. However, glucose-phosphate dehydrogenase was normal in the present case, suggesting that it is not a predisposing condition to the lung injury.

Limited global diversity of the Plasmodium vivax merozoite surface protein 4 gene

Merozoite surface proteins (MSPs) of the malaria parasites are major candidates for vaccine development targeting asexual blood stages. However, the diverse antigenic repertoire of these antigens that induce strain-specific protective immunity in human is a major challenge for vaccine design and often determines the efficacy of a vaccine. Here we further assessed the genetic diversity of Plasmodium vivax MSP4 (PvMSP4) protein using 195 parasite samples collected mostly from Thailand, Indonesia and Brazil. Overall, PvMSP4 is highly conserved with only eight amino acid substitutions. The majority of the haplotype diversity was restricted to the two short tetrapeptide repeat arrays in exon 1 and 2, respectively. Selection and neutrality tests indicated that exon 1 and the entire coding region of PvMSP4 were under purifying selection. Despite the limited nucleotide polymorphism of PvMSP4, significant genetic differentiation among the three major parasite populations was detected. Moreover, microgeographical heterogeneity was also evident in the parasite populations from different endemic areas of Thailand. (C) 2009 Elsevier B.V. All rights reserved.