James W. Kazura

Plasmodium falciparum erythrocyte invasion through glycophorin C and selection for Gerbich negativity in human populations

Geographic overlap between malaria and the occurrence of mutant hemoglobin and erythrocyte surface proteins has indicated that polymorphisms in human genes have been selected by severe malaria. Deletion of exon 3 in the glycophorin C gene (called GYPCΔex3 here) has been found in Melanesians; this alteration changes the serologic phenotype of the Gerbich (Ge) blood group system, resulting in Ge negativity. The GYPCΔex3 allele reaches a high frequency (46.5%) in coastal areas of Papua New Guinea where malaria is hyperendemic. The Plasmodium falciparum erythrocyte-binding antigen 140 (EBA140, also known as BAEBL) binds with high affinity to the surface of human erythrocytes. Here we show that the receptor for EBA140 is glycophorin C (GYPC) and that this interaction mediates a principal P. falciparum invasion pathway into human erythrocytes. EBA140 does not bind to GYPC in Ge-negative erythrocytes, nor can P. falciparum invade such cells using this invasion pathway. This provides compelling evidence that Ge negativity has arisen in Melanesian populations through natural selection by severe malaria.

Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with pfcrt polymorphism in Papua New Guinea and South America

The mechanistic basis for chloroquine resistance (CQR) in Plasmodium falciparum recently has been linked to the polymorphic gene pfcrt. Alleles associated with CQR in natural parasite isolates harbor threonine (T), as opposed to lysine (K) at amino acid 76. P. falciparum CQR strains of African and Southeast Asian origin carry pfcrt alleles encoding an amino acid haplotype of CVIET (residues 72–76), whereas most South American CQR strains studied carry an allele encoding an SVMNT haplotype; chloroquine-sensitive strains from malarious regions around the world carry a CVMNK haplotype. Upon investigating the origin of pfcrt alleles in Papua New Guinean (PNG) P. falciparum we found either the chloroquine-sensitive-associated CVMNK or CQR-associated SVMNT haplotypes previously seen in Brazilian isolates. Remarkably we did not find the CVIET haplotype observed in CQR strains from Southeast Asian regions more proximal to PNG. Further we found a previously undescribed CQR phenotype to be associated with the SVMNT haplotype from PNG and South America. This CQR phenotype is significantly less responsive to verapamil chemosensitization compared with the effect associated with the CVIET haplotype. Consistent with this, we observed that verapamil treatment of P. falciparum isolates carrying pfcrt SVMNT is associated with an attenuated increase in digestive vacuole pH relative to CVIET pfcrt-carrying isolates. These data suggest a key role for pH-dependent changes in hematin receptor concentration in the P. falciparum CQR mechanism. Our findings also suggest that P. falciparum CQR has arisen through multiple evolutionary pathways associated with pfcrt K76T.

The Role of Animal Models for Research on Severe Malaria

In light of the recent controversies over the role of animal models for research into the development of new treatments for severe malaria, particularly cerebral disease, a group of scientists came together to discuss the relative merits of a range of animal models and their overlap with the complex clinical syndromes of human disease. While it was not possible to fully resolve differences over the utility of the Plasmodium berghei ANKA model of experimental cerebral malaria, the meeting did bring the two research communities closer together to identify further work to provide information needed to validate the model and revitalise the development of other animal models displaying features of human pathology. The driving force behind this was the desire to ensure better translation of experimental findings into effective treatments for severe malaria.

Evidence That Invasion-Inhibitory Antibodies Specific for the 19-kDa Fragment of Merozoite Surface Protein-1 (MSP-119) Can Play a Protective Role against Blood-Stage Plasmodium falciparum Infection in Individuals in a Malaria Endemic Area of Africa

The C-terminal 19-kDa fragment of Plasmodium falciparum merozoite surface protein-1 (MSP-119) is a target of protective Abs against blood-stage infection and a leading candidate for inclusion in a human malaria vaccine. However, the precise role, relative importance, and mechanism of action of Abs that target this protein remain unclear. To examine the potential protective role of Abs to MSP-119 in individuals naturally exposed to malaria, we conducted a treatment time to infection study over a 10-wk period in 76 residents of a highland area of western Kenya during a malaria epidemic. These semi-immune individuals were not all equally susceptible to reinfection with P. falciparum following drug cure. Using a new neutralization assay based on transgenic P. falciparum expressing the P. chabaudi MSP-119 orthologue, individuals with high-level MSP-119-specific invasion-inhibitory Abs (>75th percentile) had a 66% reduction in the risk of blood-stage infection relative to others in the population (95% confidence interval, 3–88%). In contrast, high levels of MSP-119 IgG or IgG subclass Abs measured by enzyme immunoassay with six different recombinant MSP-119 Ags did not correlate with protection from infection. IgG Abs measured by serology and functional invasion-inhibitory activity did not correlate with each other. These findings implicate an important protective role for MSP-119-specific invasion inhibitory Abs in immunity to blood-stage P. falciparum infection, and suggest that the measurement of MSP-119 specific inhibitory Abs may serve as an accurate correlate of protection in clinical trials of MSP-1-based vaccines.

Exposure to holoendemic malaria results in elevated Epstein-Barr virus loads in children

Perennial and intense malaria transmission (holoendemic malaria) and Epstein-Barr virus (EBV) infection are 2 cofactors in the pathogenesis of endemic Burkitt lymphoma (eBL). In the present study, we compared EBV loads in children living in 2 regions of Kenya with differing malaria transmission intensities: Kisumu District, where malaria transmission is holoendemic, and Nandi District, where malaria transmission is sporadic. For comparison, blood samples were also obtained from US adults, Kenyan adults, and patients with eBL. Extraction of DNA from blood and quantification by polymerase chain reaction give an EBV load estimate that reflects the number of EBV-infected B cells. We observed a significant linear trend in mean EBV load, with the lowest EBV load detected in US adults and increasing EBV loads detected in Kenyan adults, Nandi children, Kisumu children, and patients with eBL, respectively. In addition, EBV loads were significantly higher in Kisumu children 1-4 years of age than in Nandi children of the same age. Our results support the hypothesis that repeated malaria infections in very young children modulate the persistence of EBV and increase the risk for the development of eBL.

In utero exposure to helminth and mycobacterial antigens generates cytokine responses similar to that observed in adults.

Neonates exposed to parasite antigens (Ags) in utero may develop altered fetal immunity that could affect subsequent responses to infection. We hypothesized that cord blood lymphocytes (CBL) from offspring of mothers residing in an area highly endemic for schistosomiasis, filariasis, and tuberculosis in Kenya would either fail to respond or generate a predominantly Th2-associated cytokine response to helminth and mycobacterial antigens (PPD) in vitro compared to maternal PBMC. Kenyan CBL generated helminth Ag-specific IL-5 (range 29-194 pg/ml), IL-10 (121-2,115 pg/ml), and/or IFN-gamma (78 pg/ml-10.6 ng/ml) in 26, 46, and 57% of neonates, respectively (n = 40). PPD induced IFN-gamma in 30% of Kenyan CBL (range 79-1,896 pg/ml), but little or no IL-4 or IL-5. No Ag-specific IL-4, IL-5, or IFN-gamma release was detected by CBL obtained in the United States (n = 11). Ag-driven cytokine production was primarily CD4-dependent. Cytokine responses to helminth and mycobacterial Ags by maternal PBMC mirrored that observed in neonates. CBL from helminth infected and/or PPD-sensitized mothers produced more Ag-specific cytokines compared to CBL from uninfected mothers (P < 0.05). These data demonstrate that the human fetus develops similar patterns of cytokine production observed in adults and indicates that prenatal exposure may not lead to tolerance or altered fetal immunity. .

Mathematical modelling and the control of lymphatic filariasis

The current global initiative to eliminate lymphatic filariasis represents one of the largest mass drug administration programmes ever conceived for the control of a parasitic disease. Yet, it is still not known whether the WHO-recommended primary strategy of applying annual single-dose mass chemotherapy with a combination of two drugs for 4-6 years will effectively break parasite transmission from all endemic communities. Here we review recent work on the development and application of a deterministic mathematical model of filariasis transmission, to show how models of parasite transmission will help resolve the key currently debated questions regarding the ultimate effectiveness of the global strategy to control filariasis. These critical questions include the required duration of mass treatment in different endemic areas, the optimal drug coverage required to meet control targets within prescribed timeframes, the impact and importance of adding vector control to mass chemotherapy regimens, and the likelihood of the development of drug resistance by treated worm populations. The results demonstrate the vital role that integrating these models into control programming can have in providing effective decision-support frameworks for undertaking the optimal design and monitoring of regional and global filariasis-control programmes. Operationally, the models show that the effectiveness of the strategy to achieve filariasis control will be determined by successfully addressing two key factors: the need to maintain high community treatment coverages, and the need to include vector control measures especially in areas of high endemicity.

Randomised community-based trial of annual single-dose diethylcarbamazine with or without ivermectin against Wuchereria bancrofti infection in human beings and mosquitoes

BACKGROUND WHO has targeted lymphatic filariasis for elimination. Studies of vector-parasite relations of Wuchereria bancrofti suggest that a reduction in the microfilarial reservoir by mass chemotherapy may interrupt transmission and thereby eliminate infection. However, no field data exist on the impact of chemotherapy alone on vector efficiency and transmission intensity of W bancrofti. We compared the impact of an annual community-wide single-dose treatment with diethylcarbamazine alone or with ivermectin on rate and intensity of microfilaraemia, and transmission intensity in an area of Papua New Guinea endemic for intense W bancrofti transmission. METHODS We carried out clinical and parasitological surveys in 14 communities in matched pairs. People aged 5 years or older in seven communities received randomly assigned diethylcarbamazine 6 mg/kg and people in the other seven communities received diethylcarbamazine 6 mg/kg plus ivermectin 400 micrograms/kg. We made physical examinations for hydroceles and leg oedema and investigated microfilarial densities by membrane filtration before and after treatment. We selected five communities for monthly entomological surveys between September, 1993, and September, 1995. Mosquitoes were collected in these communities by the all-night landing catch method and were individually dissected to identify rates of infection and infectiveness. FINDINGS 2219 (87.6%) of 2534 eligible people received treatment. Microfilarial rate and density had decreased 1 year after treatment in all 14 communities; this decrease was significantly higher in communities given combined therapy than in those given diethylcarbamazine alone (mean decreases 57.5% and 30.6%, respectively; p = 0.0013). Greater decreases were also seen in community-specific microfilarial intensity with combined therapy (mean reductions 91.1% and 69.8%, respectively; p = 0.0047). The rate of leg oedema was not altered, but the frequency of advanced hydroceles decreased by 47% with combined therapy and 56% with diethylcarbamazine alone. 26,641 Anopheles punctulatus mosquitoes were caught during 499 person-nights of landing catches. Exposure to infective third-stage larvae decreased in all monitored five communities. Annual transmission potential decreased by between 75.7% and 98.8% in combined-therapy communities and between 75.6% and 79.4% in communities given diethylcarbamazine alone. Transmission was almost interrupted in two communities treated with combined therapy. INTERPRETATION Annual single-dose community-wide treatment with diethylcarbamazine alone or with ivermectin is effective for the control of lymphatic filariasis in highly endemic areas, but combination therapy brings about greater decreases in rates and intensity of microfilaraemia.

Role of Cell-generated Hydrogen Peroxide in Granulocyte-mediated Killing of Schistosomula of Schistosoma mansoni In Vitro

Human as well as murine granulocytes have been shown to kill the larval stages of helminth parasites; the mechanism of this cell-mediated cytotoxicity is, however, poorly understood. The present study was designed to assess the role of peroxidative processes in killing of schistosomula of Schistosoma mansoni by human granulocytes in vitro. The rate of H(2)O(2) production by human neutrophils, eosinophils, and basophils was measured upon incubation with schistosomula alone or in the presence of specific antibody or complement. Opsonized parasites (antibody and/or complement) increased the rate of H(2)O(2) production by neutrophils, eosinophils, and basophils by respective percentages of 500, 500, and 371. The rate of H(2)O(2) release was directly related to the number of granulocytes and to the proportion of cells attached to the surface of the schistosomula. Increased hydrogen peroxide release occurred by 10 min of incubation and was demonstrable up to 16 h after addition of leukocytes to schistosomula. The primary source of this oxygen product was found to be the granulocytes adherent to the schistosomula and not those that remained unattached. Hydrogen peroxide production by neutrophils and eosinophils was quantitatively similar (schistosomula coated with antibody plus complement stimulated 5 x 10(6) neutrophils and eosinophils to release H(2)O(2) at respective rates of 0.35 and 0.40 nmol/min). Granulocyte-mediated parasite killing correlated with rate of H(2)O(2) generation; both processes were inhibited by catalase. To define further the role of oxidative metabolites, neutrophils and eosinophils of two subjects with chronic granulomatous disease were used; marked reduction of granulocyte-mediated parasite mortality was observed. Peroxidase was required for H(2)O(2)-mediated killing. Addition of the peroxidase inhibitors azide (1 mM), cyanide (1 mM), or aminotriazole (1 cM) to neutrophilschistosomula mixtures significantly reduced parasite cytotoxicity (P < 0.01); similar reduction was observed when eosinophils were used (P < 0.01). Fixation of halide (iodide) to trichloroacetic acid-precipitable protein (2.4-6.0 nmol/h per 10(7) neutrophils) was demonstrated in the presence of granulocytes, opsonins, and parasites; this process was completely inhibited by 1 mM azide. These data indicate that contact between the surfaces of human granulocytes and schistosomula results in release of cellular hydrogen peroxide and iodination. The generation of H(2)O(2) and its interaction with peroxidase appear to be crucial in effecting in vitro granulocyte-mediated parasite cytotoxicity.

Innate Immune Responses to Endosymbiotic Wolbachia Bacteria in Brugia malayi and Onchocerca volvulus Are Dependent on TLR2, TLR6, MyD88, and Mal, but Not TLR4, TRIF, or TRAM

The discovery that endosymbiotic Wolbachia bacteria play an important role in the pathophysiology of diseases caused by filarial nematodes, including lymphatic filariasis and onchocerciasis (river blindness) has transformed our approach to these disabling diseases. Because these parasites infect hundreds of millions of individuals worldwide, understanding host factors involved in the pathogenesis of filarial-induced diseases is paramount. However, the role of early innate responses to filarial and Wolbachia ligands in the development of filarial diseases has not been fully elucidated. To determine the role of TLRs, we used cell lines transfected with human TLRs and macrophages from TLR and adaptor molecule-deficient mice and evaluated macrophage recruitment in vivo. Extracts of Brugia malayi and Onchocerca volvulus, which contain Wolbachia, directly stimulated human embryonic kidney cells expressing TLR2, but not TLR3 or TLR4. Wolbachia containing filarial extracts stimulated cytokine production in macrophages from C57BL/6 and TLR4−/− mice, but not from TLR2−/− or TLR6−/− mice. Similarly, macrophages from mice deficient in adaptor molecules Toll/IL-1R domain-containing adaptor-inducing IFN-β and Toll/IL-1R domain-containing adaptor-inducing IFN-β-related adaptor molecule produced equivalent cytokines as wild-type cells, whereas responses were absent in macrophages from MyD88−/− and Toll/IL-1R domain-containing adaptor protein (TIRAP)/MyD88 adaptor-like (Mal) deficient mice. Isolated Wolbachia bacteria demonstrated similar TLR and adaptor molecule requirements. In vivo, macrophage migration to the cornea in response to filarial extracts containing Wolbachia was dependent on TLR2 but not TLR4. These results establish that the innate inflammatory pathways activated by endosymbiotic Wolbachia in B. malayi and O. volvulus filaria are dependent on TLR2-TLR6 interactions and are mediated by adaptor molecules MyD88 and TIRAP/Mal.