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Background Humanity has become largely dependent on artemisinin derivatives for both the treatment and control of malaria, with few alternatives available. A Plasmodium falciparum phenotype with delayed parasite clearance during artemisinin-based combination therapy has established in Southeast Asia, and is emerging elsewhere. Therefore, we must know how fast, and by how much, artemisinin-resistance can strengthen. Methods P. falciparum was subjected to discontinuous in vivo artemisinin drug pressure by capitalizing on a novel model that allows for long-lasting, high-parasite loads. Intravenous artesunate was administered, using either single flash-doses or a 2-day regimen, to P. falciparum-infected humanized NOD/SCID IL-2Rγ−/−immunocompromised mice, with progressive dose increments as parasites recovered. The parasite’s response to artemisinins and other available anti-malarial compounds was characterized in vivo and in vitro. Results Artemisinin resistance evolved very rapidly up to extreme, near-lethal doses of artesunate (240 mg/kg), an increase of > 3000-fold in the effective in vivo dose, far above resistance levels reported from the field. Artemisinin resistance selection was reproducible, occurring in 80% and 41% of mice treated with flash-dose and 2-day regimens, respectively, and the resistance phenotype was stable. Measuring in vitro sensitivity proved inappropriate as an early marker of resistance, as IC50 remained stable despite in vivo resistance up to 30 mg/kg (ART-S: 10.7 nM (95% CI 10.2–11.2) vs. ART-R30: 11.5 nM (6.6–16.9), F = 0.525, p = 0.47). However, when in vivo resistance strengthened further, IC50 increased 10-fold (ART-R240 100.3 nM (92.9–118.4), F = 304.8, p

Peptide vaccine strategies using Plasmodium-derived antigens have emerged as an attractive approach against malaria. However, relatively few studies have been conducted with malaria-exposed populations from non-African countries. Herein, the seroepidemiological profile against Plasmodium falciparum of naturally exposed individuals from a Brazilian malaria-endemic area against synthetic peptides derived from vaccine candidates circumsporozoite protein (CSP), liver stage antigen-1 (LSA-1), erythrocyte binding antigen-175 (EBA-175), and merozoite surface protein-3 (MSP-3) was investigated. Moreover, human leukocyte antigen (HLA)-DRB1* and HLA-DQB1* were evaluated to characterize genetic modulation of humoral responsiveness to these antigens. The study was performed using blood samples from 187 individuals living in rural malaria-endemic villages situated near Porto Velho, Rondonia State. Specific IgG and IgM antibodies and IgG subclasses were detected by enzyme-linked immunosorbent assay, and HLA-DRB1* and HLA-DQB1* low-resolution typing was performed by PCR-SSP. All four synthetic peptides were broadly recognized by naturally acquired antibodies. Regarding the IgG subclass profile, only CSP induced IgG1 and IgG3 antibodies, which is an important fact given that the acquisition of protective immunity appears to be associated with the cytophilicity of IgG1 and IgG3 antibodies. HLA-DRB1*11 and HLA-DQB1*7 had the lowest odds of responding to EBA-175. Our results showed that CSP, LSA-1, EBA, and MSP-3 are immunogenic in natural conditions of exposure and that anti-EBA antibody responses appear to be modulated by HLA class II antigens.

A major constraint in the study of Plasmodium falciparum malaria, including vaccine development, lies on the parasite's strict human host specificity and therefore the shortage of animal experimental models able to harbor human plasmodia. The best experimental models are neo-tropical primates of the genus Saimiri and Aotus, but they require splenectomy to reduce innate defenses for achieving high and consistent parasitemias, an important limitation. Clodronate-liposomes (CL) have been successfully used to deplete monocytes/macrophages in several experimental models. We investigated whether a reduction in the numbers of phagocytic cells by CL would improve the development of P. falciparum parasitemia in non-splenectomized Saimiri sciureus monkeys. Depletion of S. sciureus splenocytes after in vitro incubation with CL was quantified using anti-CD14 antibodies and flow cytometry. Non-infected and P. falciparum-infected S. sciureus were injected intravenously twice a week with either CL at either 0.5 or 1 mL (5 mg/mL) or phosphate buffered saline (PBS). Animals were monitored during infection and treated with mefloquine. After treatment and euthanasia, spleen and liver were collected for histological analysis. In vitro CL depleted S. sciureus splenic monocyte/macrophage population in a dose- and time-dependent manner. In vivo, half of P. falciparum-infected S. sciureus treated with CL 0.5 mL, and two-thirds of those treated with CL 1 mL developed high parasitemias requiring mefloquine treatment, whereas all control animals were able to self-control parasitemia without the need for antimalarial treatment. CL-treated infected S. sciureus showed a marked decrease in the degree of splenomegaly despite higher parasitemias, compared to PBS-treated animals. Histological evidence of partial monocyte/macrophage depletion, decreased hemozoin phagocytosis and decreased iron recycling was observed in both the spleen and liver of CL-treated infected S. sciureus. CL is capable of promoting higher parasitemia in P. falciparum-infected S. sciureus, associated with evidence of partial macrophage depletion in the spleen and liver. Macrophage depletion by CL is therefore a practical and viable alternative to surgical splenectomy in this experimental model.

International audience; According to the WHO, and despite reduction in mortality rates, there were an estimated 438 000 malaria deaths in 2015. Therefore new antimalarials capable of limiting organ damage are still required. We show that systemic and lung adenovirus (Ad)-mediated over-expression of trappin-2 (T-2) an antibacterial molecule with anti-inflammatory activity, increased mice survival following infection with the cerebral malaria-inducing Plasmodium berghei ANKA (PbANKA) strain. Systemically, T-2 reduced PbANKA sequestration in spleen, lung, liver and brain, associated with a decrease in pro-inflammatory cytokines (eg TNF-α in spleen and lung) and an increase in IL-10 production in the lung. Similarly, local lung instillation of Ad-T-2 resulted in a reduced organ parasite sequestration and a shift towards an anti-inflammatory/repair response, potentially implicating monocytes in the protective phenotype. Relatedly, we demonstrated in vitro that human monocytes incubated with Plasmodium falciparum-infected red blood cells (Pf-iRBCs) and IgGs from hyper-immune African human sera produced T-2 and that the latter colocalized with merozoites and inhibited Pf multiplication. This array of data argues for the first time for the potential therapeutic usefulness of this host defense peptide in human malaria patients, with the aim to limit acute lung injury and respiratory distress syndrom often observed during malaria episodes.

International audience; BACKGROUND: Substantial reductions in malaria have been reported in several African countries after distribution of insecticide-treated bednets and the use of artemisinin-based combination therapies (ACTs). Our aim was to assess the effect of these policies on malaria morbidity, mosquito populations, and asymptomatic infections in a west African rural population. METHODS: We did a longitudinal study of inhabitants of Dielmo village, Senegal, between January, 2007, and December, 2010. We monitored the inhabitants for fever during this period and we treated malaria attacks with artesunate plus amodiaquine. In July, 2008, we offered longlasting insecticide (deltamethrin)-treated nets (LLINs) to all villagers. We did monthly night collections of mosquitoes during the whole study period, and we assessed asymptomatic carriage from cross-sectional surveys. Our statistical analyses were by negative binomial regression, logistic regression, and binomial or Fisher exact test. FINDINGS: There were 464 clinical malaria attacks attributable to Plasmodium falciparum during 17 858 person-months of follow-up. The incidence density of malaria attacks averaged 5*45 (95% CI 4*90-6*05) per 100 person-months between January, 2007, and July, 2008, before the distribution of LLINs. Incidence density decreased to 0*41 (0*29-0*55) between August, 2008, and August, 2010, but increased back to 4*57 (3*54-5*82) between September and December, 2010-ie, 27-30 months after the distribution of LLINs. The rebound of malaria attacks were highest in adults and children aged 10 years or older: 45 (63%) of 71 malaria attacks recorded in 2010 compared with 126 (33%) of 384 in 2007 and 2008 (p

To investigate auto-reactive antibodies against dendrites of neurons (AAD) previously reported in cerebral malaria (CM) for their functional biological activity, a serological study was conducted in a larger cohort of patients with CM and uncomplicated falciparum malaria (UM). Sera from Thai adults with CM (n=22) and UM (n=21) were tested to determine the titers of AAD by indirect fluorescent antibody test and specific antibody responses to Plasmodium falciparum antigens by ELISA. Immunoreactivity against the dendrites of neurons was observed in 100% of sera from the cerebral malaria group as compared to 71% from the non-cerebral malaria group, and the median titer of AAD was higher in CM versus UM, though the difference did not reach significance. In contrast an opposite pattern was seen for anti-P. falciparum antibody titers, which were significantly lower among CM than among UM patients, both for IgG and IgM (p=0.024 and p=0.0033, respectively). Our results indicate that this auto-immune phenomenon induced by P. falciparum infection occurs preferentially in cerebral malaria despite lower responses in parasite-specific antibody responses.

Plasmodium falciparum merozoite surface protein 3 (MSP3), the target of antibodies that mediate parasite killing in cooperation with blood monocytes and are associated with protection in exposed populations, is a vaccine candidate under development. It belongs to a family of six structurally related genes. To optimize immunogenicity, we attempted to improve its design based on knowledge of antigenicity of various regions from the conserved C terminus of the six proteins and an analysis of the immunogenicity of “tailored” constructs. The immunogenicity studies were conducted in BALB/c and C57BL/6J mice, using MSP3 (referred to here as MSP3-1) as a model. Four constructs were designed in order to assess the effect of sequences flanking the 69-amino-acid region of MSP3-1 previously shown to be the target of biologically active antibodies. The results indicate major beneficial effects of removing (i) the subregion downstream from the 69-amino-acid sequence, since antibody titers increased by 2 orders of magnitude, and (ii) the upstream subregion which, although it defines a T-helper cell epitope, is not the target of antibodies. The construct, excluding both flanking sequences, was able to induce Th1-like responses, with a dominance of cytophilic antibodies. This led to design a multigenic construct based on these results, combining the six members of the MSP3 family. This new construction was immunogenic in mice, induced antibodies that recognized the parasite native proteins, and inhibited parasite growth in the functional antibody-dependent cellular inhibition assay, thus satisfying the preclinical criteria for a valuable vaccine candidate.

We have identified new malaria vaccine candidates through the combination of bioinformatics prediction of stable protein domains in the Plasmodium falciparum genome, chemical synthesis of polypeptides, in vitro biological functional assays, and association of an antigen-specific antibody response with protection against clinical malaria. Within the predicted open reading frame of P. falciparum hypothetical protein PFF0165c, several segments with low hydrophobic amino acid content, which are likely to be intrinsically unstructured, were identified. The synthetic peptide corresponding to one such segment (P27A) was well recognized by sera and peripheral blood mononuclear cells of adults living in different regions where malaria is endemic. High antibody titers were induced in different strains of mice and in rabbits immunized with the polypeptide formulated with different adjuvants. These antibodies recognized native epitopes in P. falciparum -infected erythrocytes, formed distinct bands in Western blots, and were inhibitory in an in vitro antibody-dependent cellular inhibition parasite-growth assay. The immunological properties of P27A, together with its low polymorphism and association with clinical protection from malaria in humans, warrant its further development as a malaria vaccine candidate.

We investigated whether anti-merozoite surface protein-1 (MSP1) block 2 antibodies mediate the monocyte-dependent antibody-mediated cellular inhibition (ADCI) of Plasmodium falciparum. This study was performed because soluble molecules have been shown to trigger ADCI and because MSP1 block 2 is released following processing and is the target of cytophilic IgG3 responses in exposed populations. We assessed human anti-MSP1 block 2 antibodies against 4 P. falciparum strains that carry the 3 main block 2 sequence alleles. These antibodies were able to inhibit in vitro growth of P. falciparum only in cooperation with human monocytes, whereas no direct inhibition was observed. However, the ADCI effect was strictly allele specific. Our findings highlight a new mechanism involving MSP1 in the protection against malaria.

Liver-stage antigen 3 (LSA-3) is a new vaccine candidate that can induce protection againstPlasmodium falciparumsporozoite challenge. Using a series of long synthetic peptides (LSP) encompassing most of the 210-kDa LSA-3 protein, a study of the antigenicity of this protein was carried out in 203 inhabitants from the villages of Dielmo (n= 143) and Ndiop (n= 60) in Senegal (the level of malaria transmission differs in these two villages). Lymphocyte responses to each individual LSA-3 peptide were recorded, some at high prevalences (up to 43%). Antibodies were also detected to each of the 20 peptides, many at high prevalence (up to 84% of responders), and were directed to both nonrepeat and repeat regions. Immune responses to LSA-3 were detectable even in individuals of less than 5 years of age and increased with age and hence exposure to malaria, although they were not directly related to the level of malaria transmission. Thus, several valuable T- and B-cell epitopes were characterized all along the LSA-3 protein, supporting the antigenicity of thisP. falciparumvaccine candidate. Finally, antibodies specific for peptide LSP10 located in a nonrepeat region of LSA-3 were found significantly associated with a lower risk of malaria attack over 1 year of daily clinical follow-up in children between the ages of 7 and 15 years, but not in older individuals.

Contains fulltext : 79604.pdf (Publisher’s version ) (Closed access) The correct selection of individuals who will benefit from iron supplements in malaria-endemic regions requires improved insight in the effects of malaria on host iron homeostasis and innovative biomarkers. We assessed sequential changes in serum hepcidin and in traditional biochemical iron status indicators during an experimental Plasmodium falciparum malaria infection with five adult volunteers. The haemoglobin content of reticulocytes (Ret-H(e)) and of mature red blood cells (RBC-H(e)) represented iron incorporation into haemoglobin. Low-density parasitaemia and its treatment induced a mild increase in interleukin (IL)-6 and serum hepcidin concentrations. Despite this only mild increase, a marked hypoferraemia with a strong increase in serum ferritin concentrations developed, which was associated with a sharp fall in Ret-H(e), while RBC-H(e) remained unchanged. The ratio of soluble transferrin receptor (sTfR) to log ferritin concentrations decreased to an average nadir of 63% of the baseline value. We concluded that even mild increases in serum hepcidin and IL-6 concentrations result in a disturbed host iron homeostasis. Serum hepcidin, Ret-H(e) and Delta-H(e) (Ret-H(e) minus RBC-H(e)) are promising biomarkers to select those individuals who will benefit from iron supplements in malaria endemic regions, while the sTfR/log ferritin ratio should be used with caution to assess iron status during malaria.

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 ADCI assay aims to measure the ability of parasite-specific antibodies, which by triggering blood monocytes, control P. falciparum parasite density. The assay relies on three easily accessible components: blood monocytes, immunoglobulins, and P. falciparum in vitro culture. Yet the reliability of results depends on the quality of the three above components, and therefore great care must be taken with each of them. We describe here different protocols for successfully carrying out the ADCI assay with emphasis on procedures and validation criteria necessary to ensure meaningful results.

The immunogenicity and efficacy of a hybrid recombinant protein derived from the N-terminal end of the glutamate-rich protein (GLURP) and the C-terminal portion of the merozoite surface protein 3 (MSP3) of Plasmodium falciparum was evaluated in Saimiri sciureus monkeys. The GLURP/MSP3 hybrid protein, expressed in Lactococcus lactis , was administered in association with alum, Montanide ISA720, or complete or incomplete Freund adjuvant (CFA/IFA) in groups of five animals each. The three formulations were shown to be immunogenic, but the one with alum was shown to be weak compared to the other two, particularly CFA/IFA, which provided very high antibody titers (enzyme-linked immunosorbent assay titers of >3,000,000 and immunofluorescence antibody test titers of 6,400). After a challenge infection with P. falciparum FUP strain, all five monkeys from the GLURP/MSP3-alum group showed a rapid increase in parasitemia, reaching 10% and were treated early. The two monkeys with the highest antibody titers in group GLURP/MSP3-Montanide ISA720 had a delay in the course of parasitemia and were treated late due to a low hematocrit. In the GLURP/MSP3-CFA/IFA group, parasitemia remained below this threshold in four of the five animals and, after it reached a peak, parasitemia started to decrease and monkeys were treated late. When all animals were grouped according to the outcome, a statistically significant association between high antibody titers and partial protection was observed. The challenge infection boosted the antibody titers, and the importance of this event for vaccine efficacy in areas where this parasite is endemic is discussed. In conclusion, these data suggest that GLURP and MSP3 can induce protection against malaria infection if antibodies are induced at properly high titers.

One potential benefit of DNA vaccines is the capacity to elicit antibody and T-cell responses against multiple antigens at the same time by mixing plasmids expressing different proteins. A possible negative effect of such mixing is interference among plasmids regarding immunogenicity. In preparation for a clinical trial, we assessed the immunogenicity of GMP-produced plasmids encoding five Plasmodium falciparum proteins, PfCSP, PfSSP2, PfEXP1, PfLSA1, and PfLSA3, given as a mixture, or alone. The mixture induced higher levels of antibodies against whole parasites than did the individual plasmids, but was associated with a decrease in antibodies to individual P. falciparum proteins. T-cell responses were in general decreased by administration of the mixture. Immune responses to individual plasmids and mixtures were generally higher in inbred mice than in outbreds. In inbred BALB/c and C57BL/6 mice, coadministration of a plasmid expressing murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), increased antibody and T-cell responses, but in outbred CD-1 mice, coadministration of mGM-CSF was associated with a decrease in antibody responses. Such variability in data from studies in different strains of mice underscores the importance of genetic background on immune response and carefully considering the goals of any preclinical studies of vaccine mixtures planned for human trials.

A longitudinal study was undertaken in Burkina Faso among 293 children aged 6 months to 9 years in order to determine the correlation between an antibody response to several individual malarial antigens and malarial infection. It was found that the presence of a positive antibody response at the beginning of the rainy season to three long synthetic peptides corresponding to Plasmodium falciparum Exp-1 101-162, MSP-3 154-249 and GLURP 801-920 but not to CSP 274-375 correlated with a statistically significant decrease in malarial infection during the ongoing transmission season. The simultaneous presence of an antibody response to more than one antigen is indicative of a lower frequency of malarial infection. This gives scientific credibility to the notion that a successful malaria vaccine should contain multiple antigens.

Plasmodium falciparum malaria is a major cause of morbidity and mortality worldwide. To evaluate the efficacy of a possible vaccine antigen against P. falciparum infection, a fusion protein, derived from P. falciparum Glutamate-rich protein (GLURP) genetically coupled to P. falciparum Merozoite surface protein 3 (MSP3) was produced in Lactococcus lactis as a secreted recombinant GLURP-MSP3 fusion protein. The hybrid protein was purified to homogeneity by ion exchange and hydrophobic-interaction chromatography and its composition was verified by MALDI MS, SDS/PAGE and Western blotting with antibodies against antigenic components of GLURP and MSP3. Mice immunized with the hybrid protein produced higher levels of both GLURP- and MSP3-specific antibodies than mice immunized with either GLURP, MSP3 or a mix of both. The protective potential of the hybrid protein was also demonstrated by in vitro parasite-growth inhibition of mouse anti-GLURP-MSP3 IgG antibodies in a monocyte-dependent manner. These results indicate that the GLURP-MSP3 hybrid could be a valuable strategy for future P. falciparum vaccine development.

We performed a longitudinal clinical and parasitological follow-up study in OoDo, a village in southeast Asia in which malaria is hyperendemic, in order to assess the association between protection against malaria attacks and antibodies to three currently evaluated vaccine candidates, merozoite surface protein 1 (MSP1), MSP3, and the 220-kDa glutamate-rich protein (GLURP) from Plasmodium falciparum. Our results showed that the levels of cytophilic immunoglobulin G3 (IgG3) antibodies against conserved regions of MSP3 and GLURP were significantly correlated with protection against clinical P. falciparum malaria. In contrast, the levels of noncytophilic IgG4 antibodies against GLURP increased with the number of malaria attacks. Furthermore, we observed a complementary effect of the MSP3- and GLURP-specific IgG3 antibodies in relation to malaria protection. In the individuals that did not respond to one of the antigens, a strong response to the other antigen was consistently detected and was associated with protection, suggesting that induction of antibodies against both MSP3 and GLURP could be important for the development of protective immunity. The complementarity of the responses to the two main targets of antibody-dependent cellular inhibition identified to date provides the first rational basis for combining these two antigens in a hybrid vaccine formulation.

The liver stage of Plasmodium spp. now appears as a relevant target of immune effectors triggered by the so-called ‘‘antisporozoite’’ vaccine. Since the monitoring of immune responses at the systemic level may not faithfully reflect the local protective mechanisms, the aim of the present work was to set up a model to study the local intra-hepatic cellular responses and to compare these with the peripheral immune responses. This was achieved by intra-portal delivery of epitopic peptides, i.e. peptides containing B and T cell epitopes, which were coated onto the surface of polystyrene microbeads. The peptide-coated beads presumably mimic the hepatic schizont, and when distinct peptides are administered separately, this method of delivery allows us to decipher the immune responses resulting in mice immunised with recombinant proteins spanning several such epitopes. Using the P. falciparum liver stage antigen-3 (LSA3) molecule, which can induce protection against a sporozoite challenge, our results show that 25-Am microbeads could easily access the liver parenchyma by intra-portal injection and were distributed evenly in the liver. Also, LSA3-derived synthetic peptides coated onto microbeads initiated specific cell recruitment within 6 h. Depending on the LSA3 peptide used, the infiltrates induced differed in size, with the strongest cell recruitment obtained using nonrepeat II peptide (NR2)-coated microbeads with a mean leukocyte number of 79 per granuloma. Immunohistological studies of liver sections revealed that, irrespective of the delivered peptide, cells infiltrating the liver towards microbeads were mainly CD3 + T lymphocytes, both CD4 + (70 to 80%) and CD8 + (20 to 30%) subtypes, macrophages and dendritic cells. Cells infiltrating the granuloma had features of activated cells, with evidence of VLA-4 cell-surface expression, and production of IFN-g and IL-4. Analysis of the peripheral B and T-cell responses in the same animals revealed that, whereas the local responses were directed mainly towards NR2 and repeat peptides (RE), the peripheral T-cell response to these peptides was weak and infrequent, although antibody production was high. D 2003 Elsevier Science B.V. All rights reserved.

The ADCI assay aims to measure the ability of parasite-specific antibodies, which by triggering blood monocytes, control P. falciparum parasite density. The assay relies on three easily accessible components: blood monocytes, immunoglobulins, and P. falciparum in vitro culture. Yet the reliability of results depends on the quality of the three above components, and therefore great care must be taken with each of them. We describe here different protocols for successfully carrying out the ADCI assay with emphasis on procedures and validation criteria necessary to ensure meaningful results.

Background One of the constraints in the development of antimalarial vaccines is the limited availability of experimental models that reliably reproduce the infection and the immunopathology observed in human malaria. The best experimental models are Saimiri and Aotus neotropical primates, but they have as important limitations, the need for splenectomy for the expression of high and consistent parasitemias. The clodronate-liposome (CL) has been used to deplete macrophages in several experimental models and we tested the hypothesis that their use in Saimiri would allow the development of consistent P. falciparum parasitemias without the need for splenectomy.

Summary Background A better understanding of the effect of malaria control interventions on vector and parasite populations, acquired immunity, and burden of the disease is needed to guide strategies to eliminate malaria from highly endemic areas. We monitored and analysed the changes in malaria epidemiology in a village community in Senegal, west Africa, over 22 years. Methods Between 1990 and 2012, we did a prospective longitudinal study of the inhabitants of Dielmo, Senegal, to identify all episodes of fever and investigate the relation between malaria host, vector, and parasite. Our study included daily medical surveillance with systematic parasite detection in individuals with fever. We measured parasite prevalence four times a year with cross-sectional surveys. We monitored malaria transmission monthly with night collection of mosquitoes. Malaria treatment changed over the years, from quinine (1990–94), to chloroquine (1995–2003), amodiaquine plus sulfadoxine-pyrimethamine (2003–06), and finally artesunate plus amodiaquine (2006–12). Insecticide-treated nets (ITNs) were introduced in 2008. Findings We monitored 776 villagers aged 0–101 years for 2 378 150 person-days of follow-up. Entomological inoculation rate ranged from 142·5 infected bites per person per year in 1990 to 482·6 in 2000, and 7·6 in 2012. Parasite prevalence in children declined from 87% in 1990 to 0·3 % in 2012. In adults, it declined from 58% to 0·3%. We recorded 23 546 fever episodes during the study, including 8243 clinical attacks caused by Plasmodium falciparum , 290 by Plasmodium malariae , and 219 by Plasmodium ovale . Three deaths were directly attributable to malaria, and two to severe adverse events of antimalarial drugs. The incidence of malaria attacks ranged from 1·50 attacks per person-year in 1990 to 2·63 in 2000, and to only 0·046 in 2012. The greatest changes were associated with the replacement of chloroquine and the introduction of ITNs. Interpretation Malaria control policies combining prompt treatment of clinical attacks and deployment of ITNs can nearly eliminate parasite carriage and greatly reduce the burden of malaria in populations exposed to intense perennial malaria transmission. The choice of drugs seems crucial. Rapid decline of clinical immunity allows rapid detection and treatment of novel infections and thus has a key role in sustaining effectiveness of combining artemisinin-based combination therapy and ITNs despite increasing pyrethroid resistance. Funding Pasteur Institutes of Dakar and Paris, Institut de Recherche pour le Developpement, and French Ministry of Cooperation.

Antibodies against three long synthetic peptides (LSPs) derived from the glutamate-rich protein (GLURP) ofPlasmodium falciparumwere analyzed in three cohorts from Liberia, Ghana, and Senegal. Two overlapping LSPs, LR67 and LR68, are derived from the relatively conserved N-terminal nonrepeat region (R0), and the third, LR70, is derived from the R2 repeat region. A high prevalence of antibody responses to each LSP was observed in all three areas of endemic infection. Levels of cytophilic immunoglobulin G (IgG) antibodies against both GLURP regions were significantly correlated with protection from clinicalP. falciparummalaria. Protected children from the Ghana cohort possessed predominantly IgG1 antibodies against the nonrepeat epitope and IgG3 antibodies against the repeat epitope. T-cell proliferation responses, studied in the cohort from Senegal, revealed that T-helper-cell epitopes were confined to the nonrepeat region. When used as immunogens, the LR67 and LR68 peptides elicited strong IgG responses in outbred mice and LR67 also induced antibodies in mice of differentH-2haplotypes, confirming the presence of T-helper-cell epitopes in these constructs. Mouse antipeptide antisera recognized parasite proteins as determined by immunofluorescence and immunoblotting. This indicates that synthetic peptides derived from relatively conserved epitopes of GLURP might serve as useful immunogens for vaccination againstP. falciparummalaria.

We have recently designed a new Plasmodium falciparum mouse model and documented its potential for the study of immune effector mechanisms. In order to determine its value for drug studies, we evaluated its response to existing antimalarial drugs compared to that observed in humans. Immunocompromised BXN (bg/bg xid/xid nu/nu) mice were infected with either the sensitive NF54 strain or the multiresistant T24 strain and then treated with chloroquine, quinine, mefloquine, or dihydroartemisinin. A parallelism was observed between previously reported human responses and P. falciparum-parasitized human red blood cell (huRBC)‐ BXN mouse responses to classical antimalarial drugs, measured in terms of speed of decrease in parasitemia and of morphological alterations of the parasites. Mice infected with the sensitive strain were successfully cured after treatment with either chloroquine or mefloquine. In contrast, mice infected with the multiresistant strain failed to be cured by chloroquine or quinine but thereafter responded to dihydroartemisinin treatment. The speed of parasite clearance and the morphological alterations induced differed for each drug and matched previously reported observations, hence stressing the relevance of the model. These data thus suggest that P. falciparum-huRBC‐BXN mice can provide a valuable in vivo system and should be included in the short list of animals that can be used for the evaluation of P. falciparum responses to drugs. In the absence of a long-awaited effective vaccine, antimalarial drugs remain the main means by which to control morbidity and mortality due to Plasmodium falciparum malaria. Although several antimalarials are available, P. falciparum has gradually developed resistance to nearly all of them. Furthermore, the prevalence and degree of resistance are increasing and the pipeline of new antimalarial compounds is drying out (26). Thus, there is an urgent need to find either new combined therapies using available compounds or to develop new antimalarial drugs to replace failing ones. However, among the various reasons for the shortage of new drugs are the small number of animal species receptive to P. falciparum and their price. Immunodeficient mice have been widely used as xenogeneic transplantation models allowing in vivo investigations of human cells and organs. Recently, Badell et al. developed a model (1) in which P. falciparum-parasitized human red blood cells (P. falciparum-huRBC) can be grafted into immunodeficient (bg/bg xid/xid nu/nu) BXN laboratory mice (P. falciparumhuRBC‐BXN). In this mouse strain, which lacks T-cell, LAKC, and T-independent B-cell functions, a main factor in the survival of P. falciparum is the concomitant decrease in innate, nonadaptive immunity. Through chemical reduction of tissue macrophages and blood leukocytes, in vivo development of medium-grade P. falciparum parasitemia can be obtained in these mice in several weeks. This new model may eventually provide a valuable tool with which to investigate the biology of this malaria parasite under in vivo conditions and therefore facilitate research in the fields of chemotherapy and vaccine development. The goal of the present work was to investigate the potential value of this new animal model for antimalarial drug studies. This could be achieved only by using those drugs whose effects in humans are well established. Immunocompromised BXN mice were infected with the drug-sensitive NF54 strain or the multidrug-resistant T24 strain and treated with chloroquine, quinine, mefloquine, or dihydroartemisinin. We compared in vivo data obtained from the P. falciparum-huRBC‐BXN mouse model with the drug sensitivity of strains determined in vitro and with the response usually observed in natural infections of humans.

The development of a malaria preerythrocytic vaccine has been greatly influenced by the observation that sterile immunity could be experimentally induced in humans by immunization with Plasmodium falciparum radiation-attenuated sporozoites (9). The critical role of liver-stage trophozoites (reviewed in reference 16) led researchers to initiate a detailed study of the antigens expressed in P. falciparum preerythrocytic stages (22). To date, scientists have identified a series of new molecules expressed at sporozoite and/or liver stage (14). Liver-stage antigen 3 (LSA3), expressed on both the sporozoite surface and in the liver forms, was found to be of particular interest. LSA3 was identified by differential screening of immune responses from protected versus nonprotected volunteers (11). A number of dominant B- and T-cell epitopes to which a high prevalence of responses is detected in individuals exposed to malaria or in LSA3-immunized animals were identified (1, 2). The vaccine potential of this molecule has been recently demonstrated in the chimpanzee model, an animal susceptible and fully receptive to P. falciparum preerythrocytic stages and whose immune system is closest to that of humans. In this model, protection against successive challenges with P. falciparum sporozoites was obtained (11). However, the use of this primate for research purposes is severely hampered by cost and ethical constraints. Several homologues of P. falciparum antigens have been identified, through a variety of immunological and molecular cross-reactivity assays, in other Plasmodium species and particularly in those infecting rodents (5, 10, 12, 13, 27, 28). The identification of structurally and functionally conserved homologues of P. falciparum proteins in rodent malaria species might help to better understand the role of these molecules in the P. falciparum parasite life cycle. This is particularly true for preerythrocytic stages, where the relative ease of obtaining sporozoites and mouse primary hepatocytes would allow more extensive investigations to be carried out. In the process of identification of LSA3, a preerythrocytic subset of P. falciparum genomic fragments (22) was used to affinity purify antibodies (Abs) from sera of individuals with hyperimmunity to malaria. These Abs were consequently checked for immunofluorescence antibody test (IFAT) cross-reactivity with rodent malaria sporozoites, and in this assay Abs against all clones coding for various fragments of LSA3 proved to be strongly reactive with Plasmodium yoelii sporozoites but not with those from Plasmodium berghei. The present study was undertaken to investigate the extent of antigenic cross-reactivity between the LSA3 molecule and its putative homologue in P. yoelii and to determine whether specific human Abs are of biological significance in the mouse model. We show that such cross-reactivity exists for human anti-LSA3 Abs, which encompass four distinct LSA3 epitopes, including both nonrepeated and repeated domains, and extend to both sporozoite and liver stages of P. yoelii. A similar result was obtained using Abs from chimpanzees immunized with LSA3. Moreover, P. yoelii, but not P. berghei, sporozoites were able to induce dose-dependent proliferation of T cells collected from one of these animals. Finally, we have established that these homologous epitopes are targets for immune responses which have a biological consequence, since LSA3-specific human Abs inhibit the invasion of P. yoelii sporozoites into mouse hepatocytes both in vitro and in vivo.

It has been frequently stressed that improved methods are needed to monitor the fast spread of drug-resistant Plasmodium falciparum parasites in endemic areas. We recently developed a colourimetric microtest, double-site enzyme-linked lactate dehydrogenase enzyme immunodetection assay (DELI), to assess drug resistance in vitro. This method, which proved highly effective under laboratory conditions, was evaluated under field conditions in 2 African areas (in Senegal and Burkina Faso) in 1997 and 1998, respectively. The sensitivities of isolates from symptomatic (n = 50) and asymptomatic individuals (n = 26) infected with P. falciparum were assessed in parallel by the new DELI-microtest and the isotopic-microtest. IC50 values of the isolates determined for chloroquine, quinine, amodiaquine and mefloquine were well correlated (r = 0.79, P < 0.001). The proportions of sensitive and resistant isolates determined using the 2 methods were similar. The DELI-microtest proved to be faster to implement than the isotopic-microtest, easier to perform, and did not require sophisticated equipment. Moreover, a larger number of isolates can be tested since parasitaemias as low as 0.005% could be reliably measured with the DELI-microtest. These initial field studies thus support the value of the DELI-microtest for large-scale drug-sensitivity monitoring.

We have recently described that sustained Plasmodium falciparum growth could be obtained in immunodeficient mice. We now report the potential of this new mouse model by assaying the effect of the passive transfer of antibodies (Abs) which in humans have had a well-established effect. Our results show that the total African adult hyperimmune immunoglobulin Gs (HI-IgGs) strongly reduce P. falciparum parasitemia similarly to that reported in humans, but only when mice are concomitantly reconstituted with human monocytes (HuMNs). In contrast, neither HI-IgGs nor HuMNs alone had any direct effect upon parasitemia. We assessed the in vivo effect of epitope-specific human Abs affinity-purified on peptides derived either from the ring erythrocyte surface antigen (RESA) or the merozoite surface protein 3 (MSP3). The inoculation of low concentrations of anti-synthetic peptide from MSP3, but not of anti-RESA Abs, consistently suppressed P. falciparum in the presence of HuMNs. Parasitemia decrease was stronger and faster than that observed using HI-IgGs and as fast as that induced by chloroquine. Our observations demonstrate that this mouse model is of great value to evaluate the protective effect of different Abs with distinct specificity in the same animal, a step hardly accessible and therefore never performed before in humans.

The antigenicity of the glutamate-rich protein (GLURP) of Plasmodium falciparum was comprehensively evaluated in epitope-mapping studies utilizing a phage display library, synthetic peptides and anti-GLURP IgG preparations previously shown to promote strong antibody-dependent cellular inhibition (ADCI) effects. We identified six major B-cell epitopes within the nonrepetitive region R0, corresponding to amino acid residues 173 to 187 (P1), 193 to 207 (P3), 216 to 229 (P4), 264 to 288 (P11), 343 to 357 (P10), and 407 to 434 (S3). Of these, four (P1, P3, P4, and S3) were frequently recognized by high-titered IgG antibodies in plasma samples from immune Liberian adults (prevalence: 29.1-45.0%). The three epitopes P1, P3, and P4 contained a common motif (seven out of nine positions are identical) and may thus constitute a family of structurally related epitopes. This leaves two distinct epitopes, one (P3) representing this new epitope family and S3 as targets for biologically active antibodies. Human IgG antibodies from single plasma samples were affinity-purified against these peptides. P3-specific IgG preparations were consistently more effective in ADCI than S3-specific IgG. Among the different GLURP epitopes, we therefore suggest that the P3 epitope is potentially the most important epitope in GLURP for the development of clinical immunity to malaria in man.

For a better definition of the polymorphic features of Plasmodium falciparum parasite populations, the polymerase chain reaction (PCR) typing technique was used to investigate the genetic diversity and complexity of parasites harbored by acute P. falciparum carriers from three yet unexplored malaria-mesoendemic areas with different transmission levels: two localities in northwestern Brazil (Ariquemes and Porto Velho) and a village in Madagascar (Ankazobe). A total of 89 DNA samples were analyzed by amplification of polymorphic domains from genes encoding merozoite surface antigens 1 and 2 (MSP-1, MSP-2) and thrombospondin-related anonymous protein (TRAP) and by hybridization with allelic-family-specific probes or random-fragment-length polymorphism (RFLP). In all three localities, extensive polymorphism was observed for each marker, but the MSP-2 central repeat was the most diverse one. Similar levels of genetic diversity, allelic frequency, and infection complexity were observed in the two Brazilian localities, although the isolates had been sampled at 2-year intervals, suggesting the stability of the infecting parasite populations presenting in these regions of the Brazilian Amazon. Unexpectedly, although the entomologic inoculation rate was at least 3 times lower in Ankazobe than in the Brazilian areas. Malagasi samples appeared more complex than the Brazilian ones. The implications of these data with regard to parasite population-dynamics studies are discussed.

We have investigated the immunogenicity in chimpanzees of twelve synthetic peptides derived from four new Plasmodium falciparum molecules expressed at pre-erythrocytic stages of the human malaria parasite. These parasite molecules were initially selected through their ability to be recognized by stage restricted human antibodies. Twelve 20- to 41-mer peptides representing potential human B- or T-cell epitopes were selected from these proteins, and synthesized. Six of these were modified by a C-terminal lipidic chain in order to re-inforce their immunogenicity. Strong B- and T-helper cell responses were induced in chimpanzees by lipopeptides injected without adjuvant and by peptides in Montanide. All twelve peptides induced CD4(+) T-cell proliferative responses, as well as the secretion of IFN-gamma (some of them at very high levels) and eleven peptides induced antibody responses. The immune responses elicited in this way were reactive with native parasite proteins, as shown by recall studies with sporozoite stage proteins, and proved to be long-lasting (up to 10 months after immunization). Our results support the strategy employed to select these four new malarial antigens and the corresponding peptides, and suggest that the immunizing formulations are both efficient and clinically acceptable.

The 19kDa, C-terminal fragment of the major surface protein of Plasmodium falciparum (PfMSP1(19)) is a candidate for inclusion in a subunit malaria vaccine. In this study, we show that PfMSP1(19)-specific antibodies, affinity purified from malaria-immune human serum, can: (i) compete with invasion-inhibitory monoclonal antibodies for binding to PfMSP1(19) and (ii) mediate inhibition of parasite growth in vitro, in the absence of complement and mononuclear cells, at physiological antibody concentrations (100 micrograms/ml). Parasites expressing either the Kl or 3D7 allele of PfMSP1(19) were equally susceptible to inhibition of merozoite invasion, indicating that the target epitopes of inhibitory antibodies are conserved or cross-reactive. These studies suggest that vaccines designed to induce antibodies to PfMSP1(19) may protect against the high levels of malaria parasitaemia which are associated with clinical disease.

To address the question of how many distinct parasites are injected when a mosquito bites, we have characterized isolates resulting most probably from a single sporozoite inoculum. We describe the direct and immediate cloning on hepatocyte feeder layers of a Thai and an African Plasmodium falciparum primary isolate and the characterization of 67 independent clones by four techniques totaling nine different markers. This led to three main conclusions: (a) both the phenotypic and genotypic markers revealed an unexpectedly large degree of diversity within the clones from a single isolate; (b) the clones are nonetheless genetically related; and (c) a single mosquito inoculum would most likely be sufficient to generate considerable isolate complexity in the absence of repeated exposure. This diversity, which has been greatly underestimated in previous studies, does not bode well for the development of successful malaria control means.

A 17-mer sequence was selected as a model to study the influence of modifications of terminal ends both on the conformational of a peptide and on its antigenicity towards naturally developing antibodies. This sequence corresponded to a tandemly repeated motif, found in a long repetitive region, with high helical propensity, of a Plasmodium falciparum liver-stage antigen (LSA-1), immunogenic in man. Our model peptide was synthesized with ionizable or non-ionizable ends, or modified in both extremities by introduction of the helix-promoting residue alpha-aminoisobutyric acid (Aib). Helical contribution, absent in the 17 amino-acid sequence possessing ionizable ends, was detectable when non-ionizable ends were introduced, and dramatically increased in the Aib-modified analogue. The presence of ionizable ends totally abolished reactivity towards human sera, otherwise detectable with the peptide possessing non-ionizable ends. While modification by Aib residues was neither detrimental nor beneficial to antigenicity in solution, it clearly resulted in an improved sensitivity of the specific antibody detection when used as solid-phase antigen in ELISA.

In Brazil, no study has been done concerning the detection of malaria parasites by polymerase chain reaction (PCR) related to the diagnosis of Plasmodium falciparum malaria. In the present report we describe a highly sensitive methodology for malaria diagnosis using a nested PCR method based on amplification of the p126 P. falciparum gene detected by simple ethidium bromide staining. The P. falciparum Palo Alto strain (culture samples) was serially diluted in blood from an uninfected donor to a final level of parasitemia corresponding to 10(-8)% and was processed for PCR amplification. In each of these dilutions a parasitological examination was performed to compare the sensitivity with that of PCR amplification. Blood samples (field samples) were obtained from 51 malarious patients with positive thick blood smears (TBS) who were living in endemic regions of the Brazilian Amazon. They corresponded to 42 P. falciparum and 9 P. vivax cases, with parasitemia levels ranging from only 16 to 20,200 parasites/microliter for P. falciparum disease and from 114 to 11,000 parasites/microliter for P. vivax malaria. We demonstrate that the use of nested PCR allows the detection of 0.005 parasites/microliter without the use of radioactive material. The use of a 1-ml sample volume and the organic DNA extraction method should be suitable in blood banks and for the evaluation of patients during and after drug treatment.

In high endemicity areas, malaria is a chronic disease: examination of blood films reveals that up to half of the population, particularly children, harbour parasites at any one given time. The parasitological status of the remainder was addressed using the polymerase chain reaction, a technique 100 to 1000 times more sensitive than microscopy, on a series of samples from Dielmo, a holoendemic area of Senegal. Two-thirds of the microscopically negative individuals were found to harbour subpatent levels of Plasmodium falciparum, suggesting that more than 90% of the exposed population at any one time, i.e. in a cross-sectional survey, are chronically infected. This also means that the range of parasite loads harboured by humans with various degrees of exposure is remarkably large, probably reflecting a large range of effectiveness of the defence mechanisms against malaria parasites, none of which is fully efficient.

A polymerase chain reaction (PCR) typing technique, based on the amplification of polymorphic regions from the merozoite surface protein 1 (MSP-1) and MSP-2 Plasmodium falciparum genes, was used to characterize parasites collected in a longitudinal study of asymptomatic carriers of malaria parasites living in two distinct epidemiologic situations. Blood samples were collected from children and adults living in the village of Dielmo, Senegal, when malaria transmission was 3-6 infective bites/week/individual. For each individual, every sample collected at two-week intervals over a period of three months showed a specific PCR pattern. Changes involved both appearance and disappearance of specific alleles. Analysis of blood samples collected at a few-days interval showed that modifications of the PCR patterns occurred rapidly. Most alleles were detected over a period of 2-3 weeks, but some alleles could be detected only for a few days. The frequent modifications of the PCR patterns indicate significant changes in allelic balance over time, and importantly, this was observed both in children and adults. These results strongly contrast with the stability of the parasite types harbored by asymptomatic individuals living in Pikine, Senegal during a period in which malaria transmission was interrupted, and therefore suggest that the rapid turnover observed in Dielmo may reflect the introduction of new parasite populations by mosquitoes.

A drug-resistance survey was conducted in the French territory of Mayotte in the Comorian Islands in the Indian Ocean where malaria is endemic. A high prevalence of resistant Plasmodium falciparum parasites was observed, not only to chloroquine (88%) and pyrimethamine (99%), but more surprisingly to quinine (17%), mefloquine (9%), and amodiaquine (24%). This leaves few treatment alternatives other than artemisine-mefloquine combinations. However, despite notification to French Health authorities three years ago, inadequate treatment (chloroquine plus sulfadoxine-pyrimethamine) is still used in this locality. Thus, people still die of malaria in this remote territory of France.

Background: The emergence and spread of Plasmodium falciparum and Plasmodium vivax resistance to available anti-malarial drugs represents a major drawback in the control of malaria and its associated morbidity and mortality. The aim of this study was to evaluate the chemoresistance profile of P. falciparum and P. vivax to commonly used anti-plasmodial drugs in a malaria-endemic area in the Brazilian Amazon. Methods: The study was carried out in Manaus (Amazonas state), in the Brazilian Amazon. A total of 88 P. falciparum and 178 P. vivax isolates was collected from 2004 to 2007. The sensitivity of P. falciparum isolates was determined to chloroquine, quinine, mefloquine and artesunate and the sensitivity of P. vivax isolates was determined to chloroquine and mefloquine, by using the colorimetric DELI test. Results: As expected, a high prevalence of P. falciparum isolates resistant to chloroquine (78.1%) was observed. The prevalence of isolates with profile of resistance or decreased sensitivity for quinine, mefloquine and artesunate was 12.7, 21.2 and 11.7%, respectively. In the case of P. vivax, the prevalence of isolates with profile of resistance for chloroquine and mefloquine was 9.8 and 28%, respectively. No differences in the frequencies of isolates with profile of resistance or geometric mean IC50s were seen when comparing the data obtained in 2004, 2005, 2006 and 2007, for all tested anti-malarials. Conclusions: The great majority of P. falciparum isolates in the Brazilian malaria-endemic area remain resistant to chloroquine, and the decreased sensitivity to quinine, mefloquine and artesunate observed in 10-20% of the isolates must be taken with concern, especially for artesunate. Plasmodium vivax isolates also showed a significant proportion of isolates with decreased sensitivity to chloroquine (first-line drug) and mainly to mefloquine. The data presented here also confirm the usefulness of the DELI test to generate results able to impact on public health policies.