Differential antibody responses to Plasmodium falciparum and P. vivax circumsporozoite proteins in a human population.

Papua New Guineans exposed to hyperendemic malaria in the Madang area showed different antibody responses to Plasmodium falciparum and Plasmodium vivax sporozoites despite comparable entomological inoculation rates. Although there was a significant trend of increasing prevalence of anti-P. falciparum circumsporozoite (CS) protein immunoglobulin G (IgG) with age, there was no significant increase in the antibody units of IgG recognizing P. falciparum CS proteins. Antibodies recognizing P. vivax CS proteins steadily increased in prevalence and antibody units with age. Significant trends of increasing prevalence of antibody responders (both IgG and IgM) with increasing splenic enlargement were found in the younger age groups for P. falciparum CS proteins but not for P. vivax CS proteins. When antibody responders were analyzed by quartiles, there was a trend of increasing antibody response with age against P. vivax CS peptide, but not for P. falciparum CS protein. There was no evidence for increasing protection against blood-stage infections with increasing antibody levels for either P. falciparum or P. vivax. Neither were any significant relationships found between entomological inoculation rates and either CS antibody prevalence or concentration among the villages studied.     

High levels of anti-phospholipid antibodies in uncomplicated and severe Plasmodium falciparum and in P. vivax malaria.

The majority (75%) of adult patients with uncomplicated Plasmodium falciparum and P. vivax malaria are positive for anti-phospholipid antibodies (aPLA) as demonstrated by ELISA using a panel of anionic and cationic phospholipids. The highest IgG and IgM binding was to the anionic phospholipids, phosphatidylserine (PS), phosphatidic acid (PA) and cardiolipin (CL), but excluding phosphatidylinositol (PI) to which only low antibody levels were found. Comparison of the mean IgG and IgM aPLA showed a trend for anti-PA > CL > PS > PC > PE > PI. Anti-PI levels were compared in two groups of African children, one group with non-severe and the other with severe (cerebral) falciparum malaria. Children with cerebral disease had significantly lower IgM anti-PI. The results are discussed with the view that serum-derived aPLA may have a role in 'anti-disease' immune responses. Their possible role in the opsonization and phagocytosis of parasitized erythrocytes and in thrombocytopenia is also considered.     

Generation of reactive oxygen species by blood monocytes in human Plasmodium falciparum and P. vivax infections

Generation of reactive oxygen radicals by peripheral blood monocytes was measured by luminol-dependent chemiluminescence in 23 P. vivax- and 7 P. falciparum-infected patients. The chemiluminescence index (CLI) was not found to be significantly higher in P. vivax-infected cases than in healthy controls. But in patients with P. falciparum infection, the CLI was significantly higher compared to controls as well as to P. vivax-infected patients. In two severe and complicated P. falciparum-infected cases, CLI was found to be higher than in mild cases. As immunosuppression is more marked in falciparum malaria than in vivax cases, the role of oxygen radical generation in immunopathology and causation of immunosuppression in falciparum malaria needs further investigation.     

Serological relationship of tumor necrosis factor-inducing exoantigens of Plasmodium falciparum and Plasmodium vivax.

Exoantigens of Plasmodium vivax-parasitized erythrocytes stimulated macrophages to secrete tumor necrosis factor, and antisera raised against the exoantigens inhibited this secretion. The antisera also inhibited the activity of Plasmodium falciparum and Plasmodium yoelii exoantigens, and conversely, antisera against the latter cross-reacted with the exoantigens of P. vivax.     

Expression and function of pvcrt-o, a Plasmodium vivax ortholog of pfcrt, in Plasmodium falciparum and Dictyostelium discoideum

Chloroquine resistance in Plasmodium vivax threatens the use of this drug as first-line treatment for millions of people infected each year worldwide. Unlike Plasmodium falciparum, in which chloroquine resistance is associated with mutations in the pfcrt gene encoding a digestive vacuole transmembrane protein, no point mutations have been associated with chloroquine resistance in the P. vivax ortholog gene, pvcrt-o (also called pvcg10). However, the question remains whether pvcrt-o can affect chloroquine response independent of mutations. Since P. vivax cannot be cultured in vitro, we used two heterologous expression systems to address this question. Results from the first system, in which chloroquine sensitive P. falciparum parasites were transformed with pvcrt-o, showed a 2.2-fold increase in chloroquine tolerance with pvcrt-o expression under a strong promoter; this effect was reversed by verapamil. In the second system, wild type pvcrt-o or a mutated form of the gene was expressed in Dictyostelium discoideum. Forms of PvCRT-o engineered to express either lysine or threonine at position 76 produced a verapamil-reversible reduction of chloroquine accumulation in this system to 60% of that in control cells. Our data support an effect of PvCRT-o on chloroquine transport and/or accumulation by P. vivax, independent of the K76T amino acid substitution.     

Evolution of the levels of soluble interleukin-2 receptors during Plasmodium falciparum and P. vivax malaria.

Increased levels of soluble interleukin-2 receptors (IL-2R) in serum were observed in both Plasmodium falciparum- and P. vivax-infected individuals compared with nonparasitemic subjects. Clinical symptoms of P. falciparum malaria were associated with higher levels of soluble IL-2R. Temporal evolution in serum of IL-2R during the course of a malaria attack mimicked the kinetics of soluble IL-2R under experimental conditions.     

Anti-lymphocytotoxic antibodies in sera of Thai adults infected with Plasmodium falciparum or Plasmodium vivax.

Because of the potential for the elimination of lymphocytes through anti-lymphocytotoxic antibodies we examined individual sera of patients infected with falciparum or vivax malaria for the presence of antibodies against normal peripheral blood mononuclear cells. In assays done at 15 degrees C, 95% of the P. falciparum patients and 98% of the P. vivax patients showed evidence for antibody activity. Activity at 37 degrees C was significantly less than that at 15 degrees C. These studies suggest that infection with malaria induces anti-lymphocytotoxic antibodies which are predominantly cold-reactive. It is possible that this phenomenon plays a role in modulating the immune response of patients toward malaria.     

Identification and characterization of the Plasmodium falciparum RhopH2 ortholog in Plasmodium vivax

Plasmodium vivax is one of the most important human malaria species that is geographically widely endemic and potentially affects a larger number of people than its more notorious cousin, Plasmodium falciparum. During invasion of red blood cells, the parasite requires the intervention of high molecular weight complex rhoptry proteins (RhopH) that are also essential for cytoadherence. PfRhopH2, a member of the RhopH multigene family, has been characterized as being crucial during P. falciparum infection. This study describes identifying and characterizing the pfrhoph2 orthologous gene in P. vivax (hereinafter named pvrhoph2). The PvRhopH2 is a 1,369-amino acid polypeptide encoded by PVX_099930 gene, for which orthologous genes have been identified in other Plasmodium species by bioinformatic approaches. Both P. falciparum and P. vivax genes contain nine introns, and there is a high degree of similarity between the deduced amino acid sequences of the two proteins. Moreover, PvRhopH2 contains a signal peptide at its N-terminus and 12 cysteines predominantly in its C-terminal half. PvRhopH2 is localized in one of the apical organelles of the merozoite, the rhoptry, and the localization pattern is similar to that of PfRhopH2 in P. falciparum. The recombinant PvRhopH2 protein is recognized by serum antibodies of patients naturally exposed to P. vivax, suggesting that PvRhopH2 is immunogenic in humans.     

Thymopoiesis,Alterations in Dendritic Cells and Tregs,and Reduced T Cell Activation in Successful Extracorporeal Photopheresis Treatment of GVHD

Journal of Clinical Immunology - Acute graft-versus-host disease (aGVHD) is a significant complication of allogeneic hematopoietic stem cell transplant (HSCT) and negatively affects T cell...     

Regulatory T cells in human geohelminth infection suppress immune responses to BCG and Plasmodium falciparum

Chronic helminth infections induce T‐cell hyporesponsiveness, which may affect immune responses to other pathogens or to vaccines. This study investigates the influence of Treg activity on proliferation and cytokine responses to BCG and Plasmodium falciparum‐parasitized RBC in Indonesian schoolchildren. Geohelminth‐infected children's in vitro T‐cell proliferation to either BCG or pRBC was reduced compared to that of uninfected children. Although the frequency of CD4⁺CD25^hiFOXP3⁺ T cells was similar regardless of infection status, the suppressive activity differed between geohelminth‐infected and geohelminth‐uninfected groups: Ag‐specific proliferative responses increased upon CD4⁺CD25^hi T‐cell depletion in geohelminth‐infected subjects only. In addition, IFN‐γ production in response to both BCG and parasitized RBC was increased after removal of CD4⁺CD25^hi T cells. These data demonstrate that geohelminth‐associated Treg influence immune responses to bystander Ag of mycobacteria and plasmodia. Geohelminth‐induced immune modulation may have important consequences for co‐endemic infections and vaccine trials.     

Identification of the optimal third generation antifolate against P. falciparum and P. vivax

Inhibitors of dihydrofolate reductase (DHFR) have been mainstays in the treatment of falciparum malaria. Resistance to one of these antifolates, pyrimethamine, is now common in Plasmodium falciparum populations. Antifolates have not traditionally been recommended for treatment of vivax malaria. However, recent studies have suggested that a third-generation antifolate, WR99210, is remarkably effective even against highly pyrimethamine-resistant parasites from both species. Two methods were used to identify a compound that is effective against quadruple mutant alleles from P. falciparum (N51I/C59R/S108N/I164L) and from Plasmodium vivax (57L/111L/117T/173F). The first was simple yeast system used to screen a panel of WR99210 analogs. The biguanide prodrug, JPC-2056, of the 2-chloro-4-trifluoromethoxy analog of WR99210 was effective against both the P. falciparum and P. vivax enzymes, and has been selected for further development. The second method compared the analogs in silico by docking them in the known structure of the P. falciparum DHFR-thymidylate synthase. The program reproduced well the position of the triazine ring, but the calculated energies of ligand binding were very similar for different compounds and therefore did not reproduce the observed trends in biological activity. The WR99210 family of molecules is flexible due to a long bridge between the triazine ring and the substituted benzene. During docking, multiple conformations were observed for the benzene ring part of the molecules in the DHFR active site, making computer-based predictions of binding energy less informative than for more rigid ligands. This flexibility is a key factor in their effectiveness against the highly mutant forms of DHFR.     

Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination

Malaria remains a major cause of morbidity and mortality in the tropics, with Plasmodium falciparum responsible for the majority of the disease burden and P. vivax being the geographically most widely distributed cause of malaria. Gametocytes are the sexual-stage parasites that infect Anopheles mosquitoes and mediate the onward transmission of the disease. Gametocytes are poorly studied despite this crucial role, but with a recent resurgence of interest in malaria elimination, the study of gametocytes is in vogue. This review highlights the current state of knowledge with regard to the development and longevity of P. falciparum and P. vivax gametocytes in the human host and the factors influencing their distribution within endemic populations. The evidence for immune responses, antimalarial drugs, and drug resistance influencing infectiousness to mosquitoes is reviewed. We discuss how the application of molecular techniques has led to the identification of submicroscopic gametocyte carriage and to a reassessment of the human infectious reservoir. These components are drawn together to show how control measures that aim to reduce malaria transmission, such as mass drug administration and a transmission-blocking vaccine, might better be deployed.     

Plasmodium falciparum Infection of Human Volunteers Activates Monocytes and CD16+ Dendritic Cells and Induces Upregulation of CD16 and CD1c Expression

Antigen-presenting cells (APCs) are key players in the induction and regulation of immune responses. In Plasmodium falciparum malaria, determination of which cells and pathways are activated in the network of APCs remains elusive. We therefore investigated the effects of a controlled human malaria infection in healthy, malaria-naive volunteers on the subset composition and activation status of dendritic cells (DCs) and monocytes. While subsets of monocytes increased in frequency during blood-stage infection, DC frequencies remained largely stable. Activation markers classically associated with peptide presentation to and priming of αβT cells, HLA-DR and CD86, were upregulated in monocytes and inflammatory CD16 myeloid DCs (mDCs) but not in the classical CD1c, BDCA2, or BDCA3 DC subsets. In addition, these activated APC subsets showed increased expression of CD1c, which is involved in glycolipid antigen presentation, and of the immune complex binding Fcγ receptor III (CD16). Our data show that P. falciparum asexual parasites do not activate classical DC subsets but instead activate mainly monocytes and inflammatory CD16 mDCs and appear to prime alternative activation pathways via induction of CD16 and/or CD1c. Changes in expression of these surface molecules might increase antigen capture and enhance glycolipid antigen presentation in addition to the classical major histocompatibility complex class II (MHC-II) peptide presentation and thereby contribute to the initiation of T-cell responses in malaria. (This study has been registered at Clinicaltrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01086917","term_id":"NCT01086917"}}NCT01086917.)     

Dendritic Cell and NK Cell Reciprocal Cross Talk Promotes Gamma Interferon-Dependent Immunity to Blood-Stage Plasmodium chabaudi AS Infection in Mice

Dendritic cells (DCs) are important accessory cells for promoting NK cell gamma interferon (IFN-γ) production in vitro in response to Plasmodium falciparum-infected red blood cells (iRBC). We investigated the requirements for reciprocal activation of DCs and NK cells leading to Th1-type innate and adaptive immunity to P. chabaudi AS infection. During the first week of infection, the uptake of iRBC by splenic CD11c⁺ DCs in resistant wild-type (WT) C57BL/6 mice was similar to that in interleukin 15^−/− (IL-15^−/−) and IL-12p40^−/− mice, which differ in the severity of P. chabaudi AS infection. DCs from infected IL-15^−/− mice expressed costimulatory molecules, produced IL-12, and promoted IFN-γ secretion by WT NK cells in vitro as efficiently as WT DCs. In contrast, DCs from infected IL-12p40^−/− mice exhibited alterations in maturation and cytokine production and were unable to induce NK cell IFN-γ production. Coculture of DCs and NK cells demonstrated that DC-mediated NK cell activation required IL-12 and, to a lesser extent, IL-2, as well as cell-cell contact. In turn, NK cells from infected WT mice enhanced DC maturation, IL-12 production, and priming of CD4⁺ T-cell proliferation and IFN-γ secretion. Infected WT mice depleted of NK cells, which exhibit increased parasitemia, had impaired DC maturation and DC-induced CD4⁺ Th1 cell priming. These findings indicate that DC-NK cell reciprocal cross talk is critical for control and rapid resolution of P. chabaudi AS infection and provide in vivo evidence for the importance of this interaction in IFN-γ-dependent immunity to malaria.     

Molecular markers for detection and differentiation of Plasmodium falciparum and Plasmodium vivax in human blood samples by pyrosequencing

PCR amplification coupled with pyrosequencing was used to measure molecular markers that could be used to detect and differentiate Plasmodium falciparum and Plasmodium vivax in human blood samples. The detection rates were in agreement with the results of Giemsa-stained film microscopy, which is the current gold standard for detection. This method provides an exciting alternative for malaria diagnosis.     

Detection of Plasmodium falciparum,P. vivax,P. ovale,and P. malariae Merozoite Surface Protein 1-p19 Antibodies in Human Malaria Patients and Experimentally Infected Nonhuman Primates

Approximately 3.2 billion people live in areas where malaria is endemic, and WHO estimates that 350 to 500 million malaria cases occur each year worldwide. This high prevalence, and the high frequency of international travel, creates significant risk for the exportation of malaria to countries where malaria is not endemic and for the introduction of malaria organisms into the blood supply. Since all four human infectious Plasmodium species have been transmitted by blood transfusion, we sought to develop an enzyme-linked immunosorbent assay (ELISA) capable of detecting antibodies elicited by infection with any of these species. The merozoite surface protein 1 (MSP1), a P. falciparum and P. vivax vaccine candidate with a well-characterized immune response, was selected for use in the assay. The MSP1 genes from P. ovale and P. malariae were cloned and sequenced (L. Birkenmeyer, A. S. Muerhoff, G. Dawson, and S. M. Desai, Am. J. Trop. Med. Hyg. 82:996-1003, 2010), and the carboxyl-terminal p19 regions of all four species were expressed in Escherichia coli. Performance results from individual p19 ELISAs were compared to those of a commercial test (Lab 21 Healthcare Malaria enzyme immunoassay [EIA]). The commercial ELISA detected all malaria patients with P. falciparum or P. vivax infections, as did the corresponding species-specific p19 ELISAs. However, the commercial ELISA detected antibodies in 0/2 and 5/8 individuals with P. malariae and P. ovale infections, respectively, while the p19 assays detected 100% of individuals with confirmed P. malariae or P. ovale infections. In experimentally infected nonhuman primates, the use of MSP1-p19 antigens from all four species resulted in the detection of antibodies within 2 to 10 weeks postinfection. Use of MSP1-p19 antigens from all four Plasmodium species in a single immunoassay would provide significantly improved efficacy compared to existing tests.More than 3.2 billion people in the world today live in areas where malaria is endemic. The World Health Organization estimates that more than 350 to 500 million malaria clinical disease episodes occur each year worldwide, with more than 1 million deaths occurring annually in sub-Saharan Africa, mostly among children under the age of 5 years (50). The combination of high disease prevalence and high frequency of international travel creates a significant risk for the exportation of malaria to countries where the disease is nonendemic. This risk is accompanied by the potential for introduction of malaria-causing organisms into the blood supplies used for transfusions. All four principal species of Plasmodium that infect humans have been transmitted via blood transfusion in the United States (36), France (4), the United Kingdom (23), and Switzerland (19). This has resulted in the implementation of donor deferral policies in many countries that restrict blood donation by those with a history of recent travel to or emigration from regions of endemicity and by those with recent cases of clinical malaria. Recent publications indicate that the prevalence of Plasmodium knowlesi, a pathogen of simian origin, in human populations in Southeast Asia (11, 12), Singapore (37), the Philippines (31), and Thailand (21) is much higher than previously believed. However, P. knowlesi malaria appears to be a zoonotic disease and to our knowledge has been not implicated in cases of transfusion-transmitted malaria in humans.The effectiveness of donor deferral programs has previously been questioned (29), and there is concern that many donors are needlessly deferred, since the rates of imported malaria are much lower than the rates of travel to areas of endemicity (17, 35). To prevent erosion of qualified donor populations, some countries have implemented antibody screening such that only individuals who are known to have been exposed to organisms causing malaria are subject to deferral of donations rather than all donors who have traveled to or lived in regions where malaria is endemic. Commercial antibody enzyme-linked immunosorbent assays (ELISAs) are currently in use (in the United Kingdom, France, and Australia), and reinstatement of questionnaire-deferred donors is being discussed in Canada and the United States (16, 24, 42). In these cases, potential donors are tested for antibodies to Plasmodium-derived antigens within several months of deferral; when the tested individuals show negative antibody results, donation is allowed.Antibodies to asexual malaria parasites (i.e., merozoites) appear within days to weeks after the invasion of erythrocytes and can persist for months or even years (14, 49). Historically, antibodies to parasite antigens have been detected using the immunofluorescence assay (IFA). This assay is not particularly sensitive or specific and is labor-intensive, requiring careful preparation of reagents. Commercially available ELISAs have been developed that use recombinant antigens or P. falciparum whole-organism lysates for detection of immunoglobulins (IgG and/or IgM, IgA) in human serum or plasma (Lab 21 Healthcare Laboratories, United Kingdom; Cellabs, Australia; DiaMed AG, Switzerland; LG Chemical Inc., Iksan, South Korea; Green Cross, Inc., Youngin, South Korea [Genedia Malaria Ab Rapid]; and Standard Diagnostics, Suwon, South Korea). These assays are typically easier to perform and exhibit higher throughput and better sensitivity and specificity than IFA (25, 42, 47), though this is not always the case (32). Some ELISAs may be better than others for detection of antibodies against all four Plasmodium species that cause malaria in humans (44). However, none of the available commercial assays currently include P. ovale- or P. malariae-derived antigens. Because these organisms have been implicated in transfusion-transmitted malaria (TTM), it would be advantageous to include antigens from these organisms in an antibody detection assay.Antigens used in some commercial ELISAs for the capture of antibodies have included vaccine candidates, since their ability to elicit antibody responses in animals and human vaccine recipients has been predetermined and naturally occurring antibodies are measured prior to vaccination. Examples of such antigens include circumsporozoite protein (CSP), apical membrane antigen 1 (AMA-1), merozoite surface protein 1 (MSP1), and, in particular, a 19-kDa C-terminal fragment of MSP1 (MSP1-p19) (22, 25, 40). Plasmodium falciparum MSP1 has been extensively studied and was one of the very earliest vaccine candidates; it elicits a protective antibody response against severe malaria, and the presence of MSP1 antibodies correlates with protective immunity (45). MSP1 is expressed as an ∼200-kDa precursor molecule linked by a glycosyl phosphatidylinositol anchor to the merozoite surface membrane. MSP1 is processed into a complex of polypeptides on the merozoite surface, including N-terminal and central regions of 82, 30, and 38 kDa, as well as the C-terminal region of 42 kDa. At the time of invasion of red blood cells, MSP1-p42 is further processed by proteolytic cleavage into a 33-kDa fragment (MSP1-p33), which is shed with the rest of the complex, and a C-terminal 19-kDa fragment (MSP1-p19). Only the C-terminal MSP1-p19 fragment remains anchored on the merozoite surface and is carried into parasitized red blood cells (RBC) (10). In monkeys, immunization with recombinant P. falciparum MSP1-p42 and P. falciparum MSP1-p19 has been shown to elicit various degrees of protection against P. falciparum challenge (15, 26). MSP1-p19 proteins from both P. falciparum and P. vivax have been proposed as vaccine candidates (18, 41, 48).By analogy to P. falciparum and P. vivax findings, one would predict that the MSP1 genes of P. ovale and P. malariae would be useful as reagents for vaccination or antibody detection. We recently cloned and expressed the MSP1-p19 proteins of P. malariae and P. ovale as recombinant antigens in Escherichia coli (2). We report here the independent evaluation of these proteins as reagents for antibody detection using sera from human malaria patients and experimentally infected nonhuman primates. In addition, a prototype immunoassay combining MSP1-p19 antigens from all four Plasmodium species was evaluated and its performance compared to a commercially available antibody test.