Origins of sequence diversity in the malaria vaccine candidate merozoite surface protein-2 (MSP-2) in Amazonian isolates of Plasmodium falciparum

The recent evolution of Plasmodium falciparum is at odds with the extensive polymorphism found in most genes coding for antigens. Here, we examined the patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2), a major malarial repetitive surface antigen. We compared the msp-2 gene sequences from closely related clones derived from sympatric parasite isolates from Brazilian Amazonia and used microsatellite typing to examine, in these same clones, the haplotype background of chromosome 2, where msp-2 is located. We found examples of msp-2 sequence rearrangements putatively created by nonreciprocal recombinational events, such as replication slippage and gene conversion, while maintaining the chromosome haplotype. We conclude that these nonreciprocal recombination events may represent a major source of antigenic diversity in MSP-2 in P. falciparum populations with low rates of classical meiotic recombination.     

Plasmodium falciparum: worldwide sequence diversity and evolution of the malaria vaccine candidate merozoite surface protein-2 (MSP-2)

We examined patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2) of Plasmodium falciparum, a major dimorphic malaria vaccine candidate antigen, by analyzing 448 msp-2 alleles from all continents. We describe several nucleotide replacements, insertion and deletion events, frameshift mutations, and proliferations of repeat units that generate the extraordinary diversity found in msp-2 alleles. We discuss the role of positive selection exerted by naturally acquired type- and variant-specific immunity in maintaining the observed levels of polymorphism and suggest that this is the most likely explanation for the significant excess of nonsynonymous nucleotide replacements found in dimorphic msp-2 domains. Hybrid sequences created by meiotic recombination between alleles of different dimorphic types were observed in few (3.1%) isolates, mostly from Africa. We found no evidence for an extremely ancient origin of allelic dimorphism at the msp-2 locus, predating P. falciparum speciation, in contrast with recent findings for other surface malarial antigens.     

Consequences of multiple infection with Plasmodium falciparum in an area of high endemicity

Most Plasmodium falciparum infections occur in partially immune hosts in highly endemic areas. In such situations, many hosts are simultaneously infected with multiple parasite genotypes, which must lead to intense competition between different parasite populations. We here summarise a series of studies of multiple infection, mostly using polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP) genotyping of the highly polymorphic msp-2 gene. These indicate that chronic infections, characteristic of the partially immune host, appear to protect against super-infecting parasites. This protection is not seen in infants. A consequence is that selection for fast-growing (virulent) parasites, occurs mainly in the youngest, immunologically na?ve, hosts. The normal situation for P. falciparum is one in which the host is partially immune, and competition between parasite genotypes in this situation is not expected to result in selection for virulence.     

Plasmodium falciparum: sequence diversity and antibody recognition of the Merozoite surface protein-2 (MSP-2) in Brazilian Amazonia

The merozoite surface protein-2 (MSP-2) of Plasmodium falciparum comprises repeats flanked by dimorphic domains defining the allelic families FC27 and IC1. Here, we examined sequence diversity at the msp-2 locus in Brazil and its impact on MSP-2 antibody recognition by local patients. Only 25 unique partial sequences of msp-2 were found in 61 isolates examined. The finding of identical msp-2 sequences in unrelated parasites, collected 6-13 years apart, suggests that no major directional selection is exerted by variant-specific immunity in this malaria-endemic area. To examine antibody cross-reactivity, recombinant polypeptides derived from locally prevalent and foreign MSP-2 variants were used in ELISA. Foreign IC1-type variants, such as 3D7 (currently tested for human vaccination), were less frequently recognized than FC27-type and local IC1-type variants. Antibodies discriminated between local and foreign IC1-type variants, but cross-recognized structurally different local IC1-type variants. The use of evolutionary models of MSP-2 is suggested to design vaccines that minimize differences between local parasites and vaccine antigens.     

Plasmodium falciparum Genotype Diversity in Artemisinin Derivatives Treatment Failure Patients along the Thai-Myanmar Border

Genetic characteristics of Plasmodium falciparum may play a role in the treatment outcome of malaria infection. We have studied the association between diversity at the merozoite surface protein-1 (msp-1), msp-2, and glutamate-rich protein (glurp) loci and the treatment outcome of uncomplicated falciparum malaria patients along the Thai-Myanmar border who were treated with artemisinin derivatives combination therapy. P. falciparum isolates were collected prior to treatment from 3 groups of patients; 50 cases of treatment failures, 50 recrudescences, and 56 successful treatments. Genotyping of the 3 polymorphic markers was analyzed by nested PCR. The distribution of msp-1 alleles was significantly different among the 3 groups of patients but not the msp-2 and glurp alleles. The allelic frequencies of K1 and MAD20 alleles of msp1 gene were higher while RO33 allele was significantly lower in the successful treatment group. Treatment failure samples had a higher median number of alleles as compared to the successful treatment group. Specific genotypes of msp-1, msp-2, and glurp were significantly associated with the treatment outcomes. Three allelic size variants were significantly higher among the isolates from the treatment failure groups, i.e., K1_270-290, 3D7_610-630, G_650-690, while 2 variants, K1_150-170, and 3D7_670-690 were significantly lower. In conclusion, the present study reports the differences in multiplicity of infection and distribution of specific alleles of msp-1, msp-2, and glurp genes in P. falciparum isolates obtained from treatment failure and successful treatment patients following artemisinin derivatives combination therapy.     

Vaccine candidate MSP-1 from Plasmodium falciparum: a redesigned 4917 bp polynucleotide enables synthesis and isolation of full-length protein from Escherichia coli and mammalian cells.

The Plasmodium falciparum malaria parasite is the causative agent of malaria tropica. Merozoites, one of the extracellular developmental stages of this parasite, expose at their surface the merozoite surface protein-1 complex (MSP-1), which results from the proteolytic processing of a 190-200 kDa precursor. MSP-1 is highly immunogenic in humans and numerous studies suggest that this protein is an effective target for a protective immune response. Although its function is unknown, there are indications that it may play a role during invasion of erythrocytes by merozoites. The parasite-derived msp-1 gene, which is approximately 5000 bp long, contains 74% AT. This high AT content has prevented stable cloning of the full-size gene in Escherichia coli and consequently its expression in heterologous systems. Here, we describe the synthesis of a 4917 bp gene encoding MSP-1 from the FCB-1 strain of P. falciparum adjusted for human codon preferences. The synthetic msp-1 gene (55% AT) was cloned, maintained and expressed in its entirety in E.coli as well as in CHO and HeLa cells. The purified protein is soluble and appears to possess native conformation because it reacts with a panel of mAbs specific for conformational epitopes. The strategy we used for synthesizing the full-length msp-1 gene was toassemble it from DNA fragments encoding all of the major proteolytic fragments normally generated at the parasite's surface. Thus, after subcloning we also obtained each of these MSP-1 processing products as hexahistidine fusion proteins in E.coli and isolated them by affinity chromatography on Ni2+agarose. The availability of defined preparations of MSP-1 and its major processing products open up new possibilities for in-depth studies at the structural and functional level of this important protein, including the exploration of MSP-1-based experimental vaccines.     

Infection of Aotus azarae boliviensis monkeys with different strains of Plasmodium vivax

Sixty-seven splenectomized Aotus azarae boliviensis were infected with strains of Plasmodium vivax from Southeast Asia (2), New Guinea (2), North Korea (1), and Central America (3). Maximum parasitemias varied among the different strains, with the mean maximum parasitemia for the primary infection period being 16,200 per mm3. Animals previously infected with Plasmodium falciparum and Plasmodium malariae produced maximum parasitemias of 30,200 and 11,900 per mm3, respectively. Gametocytes infective to Anopheles freeborni mosquitoes were produced with 7 of the 8 strains examined.     

The relationship between reported fever and Plasmodium falciparum infection in African children

Background

The genetic diversity of Plasmodium falciparum has been extensively studied in various parts of the world. However, limited data are available from Pakistan. This study aimed to establish molecular characterization of P. falciparum field isolates in Pakistan measured with two highly polymorphic genetic markers, i.e. the merozoite surface protein 1 (msp-1)and 2 (msp-2).

Methods

Between October 2005 and October 2007, 244 blood samples from patients with symptomatic blood-slide confirmed P. falciparum mono-infections attending the Aga Khan University Hospital, Karachi, or its collection units located in Sindh and Baluchistan provinces, Pakistan were collected. The genetic diversity of P. falciparum was analysed by length polymorphism following gel electrophoresis of DNA products from nested polymerase chain reactions (PCR) targeting block 2 of msp-1 and block 3 of msp-2, including their respective allelic families KI, MAD 20, RO33, and FC27, 3D7/IC.

Results

A total of 238/244 (98%) patients had a positive PCR outcome in at least one genetic marker; the remaining six were excluded from analysis. A majority of patients had monoclonal infections. Only 56/231 (24%) and 51/236 (22%) carried multiple P. falciparum genotypes in msp-1 and msp-2, respectively. The estimated total number of genotypes was 25 msp-1 (12 KI; 8 MAD20; 5 RO33) and 33 msp-2 (14 FC27; 19 3D7/IC).

Conclusions

This is the first report on molecular characterization of P. falciparum field isolates in Pakistan with regards to multiplicity of infection. The genetic diversity and allelic distribution found in this study is similar to previous reports from India and Southeast Asian countries with low malaria endemicity.     

A murine model of falciparum-malaria by in vivo selection of competent strains in non-myelodepleted mice engrafted with human erythrocytes

To counter the global threat caused by Plasmodium falciparum malaria, new drugs and vaccines are urgently needed. However, there are no practical animal models because P. falciparum infects human erythrocytes almost exclusively. Here we describe a reliable falciparum murine model of malaria by generating strains of P. falciparum in vivo that can infect immunodeficient mice engrafted with human erythrocytes. We infected NOD(scid/beta2m-/-) mice engrafted with human erythrocytes with P. falciparum obtained from in vitro cultures. After apparent clearance, we obtained isolates of P. falciparum able to grow in peripheral blood of engrafted NOD(scid/beta2m-/-) mice. Of the isolates obtained, we expanded in vivo and established the isolate Pf3D7(0087/N9) as a reference strain for model development. Pf3D7(0087/N9) caused productive persistent infections in 100% of engrafted mice infected intravenously. The infection caused a relative anemia due to selective elimination of human erythrocytes by a mechanism dependent on parasite density in peripheral blood. Using this model, we implemented and validated a reproducible assay of antimalarial activity useful for drug discovery. Thus, our results demonstrate that P. falciparum contains clones able to grow reproducibly in mice engrafted with human erythrocytes without the use of myeloablative methods.     

Interactions between merozoite surface proteins 1, 6, and 7 of the malaria parasite Plasmodium falciparum

Merozoites of the malaria parasite Plasmodium falciparum expose at their surface a large multiprotein complex, composed of proteolytically processed, noncovalently associated products of at least three genes, msp-1, msp-6, and msp-7. During invasion of erythrocytes, this complex is shed from the surface except for a small glycosylphosphatidylinositol-anchored portion originating from MSP-1. The proteolytic cleavage separating the C-terminal portion of MSP-1 is required for successful invasion. Little is known about the structure and function of the abundant and essential multipartite complex. Using heterologously produced MSP-1, MSP-6, and MSP-7 in precursor and with the exception of MSP-7 in processed form, we have studied in vitro the complex formation between the different proteins to identify the interaction partners within the complex. Both MSP-6(36) and MSP-7 bind only to MSP-1 subunits that are shed, but although MSP-6(36) contacts just subunit p38, MSP-7 interacts with p83, p30, and p38. The intact C-terminal region of MSP-6 is required for the association with p38 as well as for its multimerization into tetramers. Furthermore, our data suggest that only the processed form and not the precursor form of MSP-1 interacts with MSP-6(36). MSP-6- as well as MSP-7-specific rabbit antibodies inhibit parasite multiplication in vitro as shown previously for antibodies directed against MSP-1. Our findings raise interesting questions with regard to proteolysis-mediated mechanisms of maturation of the MSP-1-MSP-6-MSP-7 complex and to the mode by which antibodies directed against this complex interfere with parasite multiplication.     

The course of infections and pathology in immunomodulated NOD/LtSz-SCID mice inoculated with Plasmodium falciparum laboratory lines and clinical isolates

Human chimeras are potentially invaluable models for hemoprotozoan parasites such as Plasmodium falciparum. The work presented assesses the susceptibility of immunomodulated NOD/LtSz-SCID mice to genetically distinct P. falciparum parasites. To this end, mice grafted with human erythrocytes were inoculated with two P. falciparum laboratory lines, 3D7 and Dd2 and four clinical isolates, ISCIII-230, ISCIII-231, ISCIII-381 and ISCIII-399. The results showed that, without a previous period of parasite adaptation, 100% of the inoculated mice developed an infection, generally self-limited, though some mice died. The parasitemias ranged from 0.05 to 8% and lasted an average of 19 days (15-26 days) depending on the line or isolate studied. Sexual forms of different maturity, stage II-IV and mature gametocytes were observed in the peripheral blood of mice in 22, 50, 25, 72 and 80% of the mice infected with Dd2, ISCIII-399, ISCIII-230, ISCIII-231 and ISCIII-381 isolates, respectively. The study of the clinical symptoms, the haematological parameters and the histopathological changes in the infected mice showed that most of the malaria features were present in the infected mice except that the sequestration of infected erythrocytes was absent or at most a minor phenomenon, as also indicated by the presence of mature forms of the parasites in the peripheral blood. This study shows that the human chimeras allow the complete asexual and sexual erythrocytic cycle of different P. falciparum lines and clinical isolates to be observed in vivo. It opens a new way to investigate any parasite population in terms of infectivity, transmission, and drug resistance.     

A Revised Timeline for the Origin of Plasmodium falciparum as a Human Pathogen

While Plasmodium falciparum is known to have had a strong effect on human evolution, the time period when P. falciparum first infected ancestors of modern humans has remained uncertain. Recent advances demonstrated that P. falciparum evolved from ancestors of gorilla parasites via host switching. Here, we estimate the range of dates during which this host switch may have occurred. DNA sequences of portions of the mitochondrial cytochrome b gene obtained from gorilla parasites closely related to human P. falciparum were aligned and compared against similar sequences from human P. falciparum. Time estimates were calculated by applying a previously established parasite cytochrome b gene mutation rate (0.012 mutations per site per million years) and by modeling uncertainty in a Monte-Carlo simulation. We estimate a 95% confidence interval for when P. falciparum first infected ancestors of modern humans to be 112,000 and 1,036,000 years ago (median estimate, 365,000 years ago). This confidence interval suggests that P. falciparum first infected human ancestors much more recently than the previous recognized estimate of 2.5 million years ago. The revised estimate may inform our understanding of certain aspects of human-malaria co-evolution. For example, this revised date suggests a closer relationship between the entry of P. falciparum in humans and the appearance of many red blood cell polymorphisms considered to be genetic adaptations to malaria. In addition, the confidence interval lies within the timeframe dating the dawn of Homo sapiens, suggesting that P. falciparum may have undergone host switching as a Plasmodia adaptation specific for our species.     

A comparison of Anopheles gambiae and Plasmodium falciparum genetic structure over space and time

Population genetic structure and subdivision are key factors affecting the evolution of organisms. In this study, we analysed and compared the population genetic structure of the malaria parasite Plasmodium falciparum and its mosquito vector Anopheles gambiae over space and time in the Nianza Province, near Victoria Lake in Kenya. The parasites were collected from mosquitoes caught in six villages separated by up to 68 km in 2002 and 2003. A total of 545 oocysts were dissected from 122 infected mosquitoes and genotyped at seven microsatellite markers. Five hundred and forty-seven mosquitoes, both infected and uninfected, were genotyped at eight microsatellites. For the parasite and the vector, the analysis revealed no (or very little) genetic differentiation among villages. This may be explained by high local population sizes for the parasite and the mosquito. The small level of genetic differentiation observed between populations may explain the speed at which antimalarial drug resistance and insecticide resistance spread into the African continent.     

Interrupting malaria transmission: quantifying the impact of interventions in regions of low to moderate transmission

Malaria has been eliminated from over 40 countries with an additional 39 currently planning for, or committed to, elimination. Information on the likely impact of available interventions, and the required time, is urgently needed to help plan resource allocation. Mathematical modelling has been used to investigate the impact of various interventions; the strength of the conclusions is boosted when several models with differing formulation produce similar data. Here we predict by using an individual-based stochastic simulation model of seasonal Plasmodium falciparum transmission that transmission can be interrupted and parasite reintroductions controlled in villages of 1,000 individuals where the entomological inoculation rate is <7 infectious bites per person per year using chemotherapy and bed net strategies. Above this transmission intensity bed nets and symptomatic treatment alone were not sufficient to interrupt transmission and control the importation of malaria for at least 150 days. Our model results suggest that 1) stochastic events impact the likelihood of successfully interrupting transmission with large variability in the times required, 2) the relative reduction in morbidity caused by the interventions were age-group specific, changing over time, and 3) the post-intervention changes in morbidity were larger than the corresponding impact on transmission. These results generally agree with the conclusions from previously published models. However the model also predicted changes in parasite population structure as a result of improved treatment of symptomatic individuals; the survival probability of introduced parasites reduced leading to an increase in the prevalence of sub-patent infections in semi-immune individuals. This novel finding requires further investigation in the field because, if confirmed, such a change would have a negative impact on attempts to eliminate the disease from areas of moderate transmission.     

Genetic Diversity and Selection in Three Plasmodium vivax Merozoite Surface Protein 7 (Pvmsp-7) Genes in a Colombian Population

A completely effective vaccine for malaria (one of the major infectious diseases worldwide) is not yet available; different membrane proteins involved in parasite-host interactions have been proposed as candidates for designing it. It has been found that proteins encoded by the merozoite surface protein (msp)-7 multigene family are antibody targets in natural infection; the nucleotide diversity of three Pvmsp-7 genes was thus analyzed in a Colombian parasite population. By contrast with P. falciparum msp-7 loci and ancestral P. vivax msp-7 genes, specie-specific duplicates of the latter specie display high genetic variability, generated by single nucleotide polymorphisms, repeat regions, and recombination. At least three major allele types are present in Pvmsp-7C, Pvmsp-7H and Pvmsp-7I and positive selection seems to be operating on the central region of these msp-7 genes. Although this region has high genetic polymorphism, the C-terminus (Pfam domain ID: PF12948) is conserved and could be an important candidate when designing a subunit-based antimalarial vaccine.     

Detection of Plasmodium sporozoites in mosquitoes by polymerase chain reaction and oligonucleotide rDNA probe, without dissection of the salivary glands

Dried Anopheles gambiae mosquito head + thorax portions, infected with Plasmodium falciparum sporozoites, were processed by the polymerase chain reaction. The PCR product was hybridized to an oligonucleotide probe (known as 114R or AW34) diagnostic for Plasmodium . The detection level by autoradiography was ten sporozoites per mosquito. Head + thorax of mosquitoes that contained mature P.falciparum oocysts, without sporozoites, gave no positive signal, indicating that the test detects only infective mosquitoes. This test can be applied to wild mosquito specimens collected, prepared and processed at different time intervals. The technique is convenient, highly sensitive, and could be used with a non-radioactive detection system and specific probes to differentiate Plasmodium spp.     

Moderate transmission but high prevalence of malaria in Madagascar

Malaria transmission remains poorly documented in areas of low transmission. A study has been carried out over two consecutive years in Analamiranga, a village located at an altitude of 885m on the western edge of the Malagasy highlands, with the aim of generating and updating malariometric indexes for both mosquitoes and schoolchildren. In this village, no vector control measures were performed during the study period nor during previous decades. Mosquitoes were collected monthly when landing on human volunteers and in various resting-places. Blood samples were taken every 3 months from schoolchildren aged 6-12 years and microscopically examined. Of 7,480 mosquitoes collected on human subjects, 5,790 were anophelines. Ten anopheline species were represented and three of these, Anopheles funestus, Anopheles arabiensis and Anopheles mascarensis, accounted for 59.2% of the collection. Of these three species 4,640 were also collected in resting places. The proportion of mosquitoes fed on bovids was high; conversely, the anthropophilic rate (mosquitoes fed on human beings) was especially low: 31%, 7% and 1%, respectively, for A. funestus, A. arabiensis and A. mascarensis. The only confirmed malaria vector was A. funestus with a low sporozoite index (of 6,830 A. funestus, five were positive for Plasmodium falciparum and four for Plasmodium vivax). The annual entomological inoculation rate (number of bites of infected anophelines per adult person) was estimated at 2.49 with low variation over the 2 years. Overall, 909 thick blood smears were tested from blood samples taken from schoolchildren with 30.3% being malaria-positive. The four Plasmodium species infecting human subjects were detected in the following proportions: P. falciparum 78.9%, P. vivax 19.4%, Plasmodium malariae 1.0% and Plasmodium ovale 0.7%. The proportions of children who were infected with any Plasmodium ranged from 10.7% in February to 51.0% in September. Parasitemic children with fever (axillary temperature >37.5 degrees C) accounted for 16.4% of the children sampled. This study demonstrates that there are substantial parasitological consequences of even a relatively low entomological transmission and also recommends including exterior resting-places of mosquitoes in future spraying campaigns in the highlands of Madagascar.     

Malaria diagnosis by enzyme-linked immunosorbent assays.

Enzyme-linked immunosorbent assays (ELISA) for detecting malarial parasites in blood were tested on uninfected monkeys and in monkeys infected with Plasmodium falciparum. A double antibody sandwich ELISA detected one malarial cell per 10(3) uninfected cells, and an inhibition ELISA detected on infected cell per 10(4) uninfected erythrocytes. These methods are not yet as sensitive as conventional blood-film examinations, in which a well-trained microscopist might be expected to detect one malarial cell per 10(6) erythrocytes. Nevertheless, ELISA offers an objective means of detecting malaria and is particularly advantageous since up to 100 samples may be processed simultaneously.     

Mortality associated with HIV-1 infection over five years in a rural Ugandan population: cohort study.

OBJECTIVE: To assess the impact of HIV-1 infection on mortality over five years in a rural Ugandan population. DESIGN: Longitudinal cohort study followed up annually by a house to house census and medical survey. SETTING: Rural population in south west Uganda. SUBJECTS: About 10,000 people from 15 villages who were enrolled in 1989-90 or later. MAIN OUTCOME MEASURES: Number of deaths from all causes, death rates, mortality fraction attributable to HIV-1 infection. RESULTS: Of 9777 people resident in the study area in 1989-90, 8833 (90%) had an unambiguous result on testing for HIV-1 antibody; throughout the period of follow up adult seroprevalence was about 8%. During 35,083 person years of follow up, 459 deaths occurred, 273 in seronegative subjects and 186 in seropositive subjects, corresponding to standardised death rates of 8.1 and 129.3 per 1000 person years. Standardised death rates for adults were 10.4 (95% confidence interval 9.0 to 11.8) and 114.0 (93.2 to 134.8) per 1000 person years respectively. The mortality fraction attributable to HIV-1 infection was 41% for adults and was in excess of 70% for men aged 25-44 and women aged 20-44 years. Median survival from time of enrollment was less than three years in subjects aged 55 years or more who were infected with HIV-1. Life expectancy from birth in the total population resident at any time was estimated to be 42.5 years (41.4 years in men; 43.5 years in women), which compares with 58.3 years (56.5 years in men; 60.5 years in women) in people known to be seronegative. CONCLUSIONS: These data confirm that in a rural African population HIV-1 infection is associated with high death rates and a substantial reduction in life expectancy.