Quantification of Plasmodium falciparum gametocytes in differential stages of development by quantitative nucleic acid sequence-based amplification

Two quantitative nucleic acid sequence-based amplification assays (QT-NASBA) based on Pfs16 and Pfs25, have been developed to quantify sexual stage commitment and mature gametocytes of Plasmodium falciparum. Pfs16 mRNA is expressed in all sexual forms including sexually committed ring stages while expression of Pfs25 mRNA is restricted to late stage gametocytes. Both assays showed a sensitivity of one sexual stage parasite/microl of blood. Blood samples from experimentally infected non-immune human volunteers were tested for Plasmodium falciparum by standard microscopy, a previously developed asexual 18S rRNA QT-NASBA, Pfs16 and Pfs25 mRNA QT-NASBA. Pfs16 QT-NASBA was positive in 9 out of 10 volunteers within 48 h after first detection of 18S rRNA, mostly before or at the day of positive microscopy. In contrast, the Pfs25 mRNA QT-NASBA was negative during the 28 days of follow-up, but consistently positive in gametocyte samples from naturally infected Kenyan patients. These data suggest that sexual stage commitment can occur early in the blood-stage infection without successful maturation into infectious gametocytes. In conclusion, Pfs16 and Pfs25 QT-NASBA assays in combination with a previously developed asexual stage QT-NASBA allow for the separate quantification of all developmental stages present in the circulation. The application of sexual stage QT-NASBA assays may contribute to a better understanding of the biology and epidemiology of malaria transmission.     

Immunity against sexual stage Plasmodium falciparum and Plasmodium vivax parasites

The efficient spread of malaria from infected humans to mosquitoes is a major challenge for malaria elimination initiatives. Gametocytes are the only Plasmodium life stage infectious to mosquitoes. Here, we summarize evidence for naturally acquired anti-gametocyte immunity and the current state of transmission blocking vaccines (TBV). Although gametocytes are intra-erythrocytic when present in infected humans, developing Plasmodium falciparum gametocytes may express proteins on the surface of red blood cells that elicit immune responses in naturally exposed individuals. This immune response may reduce the burden of circulating gametocytes. For both P. falciparum and Plasmodium vivax, there is a solid evidence that antibodies against antigens present on the gametocyte surface, when co-ingested with gametocytes, can influence transmission to mosquitoes. Transmission reducing immunity, reducing the burden of infection in mosquitoes, is a well-acknowledged but poorly quantified phenomenon that forms the basis for the development of TBV. Transmission enhancing immunity, increasing the likelihood or intensity of transmission to mosquitoes, is more speculative in nature but is convincingly demonstrated for P. vivax. With the increased interest in malaria elimination, TBV and monoclonal antibodies have moved to the center stage of malaria vaccine development. Methodologies to prioritize and evaluate products are urgently needed.     

Plasmodium falciparum infection elicits both variant-specific and cross-reactive antibodies against variant surface antigens

Naturally acquired antibodies to Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1), the variant surface antigens expressed on the surface of infected erythrocytes, are thought to play a role in protection against P. falciparum malaria. Here, we have studied the development of antibodies to PfEMP-1 in adult malaria patients living in Rourkela, India, an area with a low malaria transmission rate, and prevalence of antibodies to PfEMP-1 in residents of San Dulakudar, India, a village in which P. falciparum malaria is hyperendemic. Convalescent-phase sera from adult malaria patients from Rourkela agglutinate homologous P. falciparum isolates as well as some heterologous isolates, suggesting that they develop partially cross-reactive antibodies to PfEMP-1 following infection. Adult sera from San Dulakudar agglutinate diverse P. falciparum isolates, suggesting that they have antibodies with wide recognition of diverse PfEMP-1. Mixed-agglutination assays using pairs of P. falciparum isolates confirm the presence of both variant-specific and partially cross-reactive antibodies in convalescent-phase sera from Rourkela and adult sera from San Dulakudar. Analysis of PfEMP-1 sequences suggests a molecular basis for the observed cross-reactivity.     

CD36-mediated nonopsonic phagocytosis of erythrocytes infected with stage I and IIA gametocytes of Plasmodium falciparum

Gametocytes, the sexual stages of malaria parasites (Plasmodium spp.) that are transmissible to mosquitoes, have been the focus of much recent research as potential targets for novel drug and vaccine therapies. However, little is known about the host clearance of gametocyte-infected erythrocytes (GEs). Using a number of experimental strategies, we found that the scavenger receptor CD36 mediates the uptake of nonopsonized erythrocytes infected with stage I and IIA gametocytes of Plasmodium falciparum by monocytes and culture-derived macrophages (Mphis). Light microscopy and immunofluorescence assays revealed that stage I and IIA gametocytes were readily internalized by monocytes and Mphis. Pretreating monocytes and Mphis with a monoclonal antibody that blocked CD36 resulted in a significant reduction in phagocytosis, as did treating GEs with low concentrations of trypsin to remove P. falciparum erythrocyte membrane protein 1 (PfEMP-1), a parasite ligand for CD36. Pretreating monocytes and Mphis with peroxisome proliferator-activated receptor gamma-retinoid X receptor agonists, which specifically upregulate CD36, resulted in a significant increase in the phagocytosis of GEs. Murine CD36 on mouse Mphis also mediated the phagocytosis of P. falciparum stage I and IIA gametocytes, as determined by receptor blockade with anti-murine CD36 monoclonal antibodies and the lack of uptake by CD36-null Mphis. These results indicate that phagocytosis of stage I and IIA gametocytes by monocytes and Mphis appears to be mediated to a large extent by the interaction of PfEMP-1 and CD36, suggesting that CD36 may play a role in innate clearance of these early sexual stages.     

Impact of mosquito bites on asexual parasite density and gametocyte prevalence in asymptomatic chronic Plasmodium falciparum infections and correlation with IgE and IgG titers

An immunomodulatory role of arthropod saliva has been well documented, but evidence for an effect on Plasmodium sp. infectiousness remains controversial. Mosquito saliva may orient the immune response toward a Th2 profile, thereby priming a Th2 response against subsequent antigens, including Plasmodium. Orientation toward a Th1 versus a Th2 profile promotes IgG and IgE proliferation, respectively, where the former is crucial for the development of an efficient antiparasite immune response. Here we assessed the direct effect of mosquito bites on the density of Plasmodium falciparum asexual parasites and the prevalence of gametocytes in chronic, asymptomatic infections in a longitudinal cohort study of seasonal transmission. We additionally correlated these parasitological measures with IgE and IgG antiparasite and anti-salivary gland extract titers. The mosquito biting density was positively correlated with the asexual parasite density but not asexual parasite prevalence and was negatively correlated with gametocyte prevalence. Individual anti-salivary gland IgE titers were also negatively correlated with gametocyte carriage and were strongly positively correlated with antiparasite IgE titers, consistent with the hypothesis that mosquito bites predispose individuals to develop an IgE antiparasite response. We provide evidence that mosquito bites have an impact on asymptomatic infections and differentially so for the production of asexual and sexual parasites. An increased research focus on the immunological impact of mosquito bites during asymptomatic infections is warranted, to establish whether strategies targeting the immune response to saliva can reduce the duration of infection and the onward transmission of the parasite.     

The C-terminal segment of the cysteine-rich interdomain of Plasmodium falciparum erythrocyte membrane protein 1 determines CD36 binding and elicits antibodies that inhibit adhesion of parasite-infected erythrocytes

Attachment of erythrocytes infected by Plasmodium falciparum to receptors of the microvasculature is a major contributor to the pathology and morbidity associated with malaria. Adhesion is mediated by the P. falciparum erythrocyte membrane protein 1 (PfEMP-1), which is expressed at the surface of infected erythrocytes and is linked to both antigenic variation and cytoadherence. PfEMP-1 contains multiple adhesive modules, including the Duffy binding-like domain and the cysteine-rich interdomain region (CIDR). The interaction between CIDRalpha and CD36 promotes stable adherence of parasitized erythrocytes to endothelial cells. Here we show that a segment within the C-terminal region of CIDRalpha determines CD36 binding specificity. Antibodies raised against this segment can specifically block the adhesion to CD36 of erythrocytes infected with various parasite strains. Thus, small regions of PfEMP-1 that determine binding specificity could form suitable components of an antisequestration malaria vaccine effective against different parasite strains.     

Strategic use of antimalarial drugs that block falciparum malaria parasite transmission to mosquitoes to achieve local malaria elimination

The ultimate aim of malaria chemotherapy is not only to treat symptomatic infection but also to reduce transmission potential. With the absence of clinically proven vaccines, drug-mediated blocking of malaria transmission gains growing interest in the research agenda for malaria control and elimination. In addition to the limited arsenal of antimalarials available, the situation is further complicated by the fact that most commonly used antimalarials are being extensively resisted by the parasite and do not assist in blocking its transmission to vectors. Most antimalarials do not exhibit gametocytocidal and/ or sporontocidal activity against the sexual stages of Plasmodium falciparum but may even enhance gametocytogenesis and gametocyte transmissibility. Artemisinin derivatives and 8-aminoquinolines are useful transmission-blocking antimalarials whose optimal actions are on different stages of gametocytes. Transmission control interventions that include gametocytocides covering the spectrum of gametocyte development should be used to reduce and, if possible, stop transmission and infectivity of gametocytes to mosquitoes. Potent gametocytocidal drugs could also help deter the spread of antimalarial drug resistance. Novel proof-of-concept compounds with gametocytocidal activity, such as trioxaquines, synthetic endoperoxides, and spiroindolone, should be further tested for possible clinical utility before investigating the possibility of integrating them in transmission-reducing interventions. Strategic use of potent gametocytocides at appropriate timing with artemisinin-based combination therapies should be given attention, at least, in the short run. This review highlights the role that antimalarials could play in blocking gametocyte transmission and infectivity to mosquitoes and, hence, in reducing the potential of falciparum malaria transmissibility and drug resistance spread.     

Superoxide-dependent and -independent pathways are involved in the transmission blocking of malaria

Superoxide plays a crucial role in innate immunity to various pathogens. We examined the role of superoxides in the transmission of malaria using gp91phox knockout (X-CGD) mice that lack the ability to produce superoxide. Mosquitoes that fed on X-CGD mice infected intraperitoneally with Plasmodium berghei NK65 ANKA formed more oocysts than did those that fed on control mice at any day after infection. The number of oocysts peaked on day 5 post-infection in X-CGD and control mice and then decreased significantly after day 5 post-infection. However, on day 7 post-infection, the infectivity of gametocytes in X-CGD mice was significantly higher than that in control mice. These results show that two pathways, superoxide-dependent and -independent, are involved in the host systems regulating the transmission of malaria and inhibiting gametocyte development.     

Sex- and stage-specific reporter gene expression in Plasmodium falciparum

For malaria transmission, Plasmodium parasites must successfully complete gametocytogenesis in the vertebrate host. Differentiation into mature male or female Plasmodium falciparum gametocytes takes 9-12 days as the parasites pass through five distinct morphologic stages (I-V). To evaluate the signals controlling the initiation of stage- and/or sex-specific expression, reporter constructs containing the 5'-flanking regions (FR) of seven genes with distinct expression patterns through gametogenesis were developed. The regulatory information present in the 5'-FR of each selected gene was found to be sufficient to drive appropriate sex- and stage-specific reporter gene expression. The transformed parasite lines also provide in vivo markers to identify gametocytes at specific stages, including a subpopulation of schizonts that express early gametocyte markers.     

2-Cys Peroxiredoxin TPx-1 is involved in gametocyte development in Plasmodium berghei

Peroxiredoxins (Prxs) constitute a ubiquitous family of antioxidant enzymes involved in diverse cellular functions including cell proliferation and differentiation. To investigate the physiologic role of typical 2-Cys Prx in malaria parasites (TPx-1), we disrupted this gene in the rodent malaria parasite Plasmodium berghei (pbtpx-1). The gene-disrupted parasite (Prx KO) developed normally in mouse erythrocytes and multiplied at a rate similar to that of the parent strain (WT) during the experimental period. The normal growth rate was not altered after 10 passages, and the level of 8-hydroxy-2'-deoxyguanosine, which accumulates in the parasite genome during the cell cycle, was similar between Prx KO and WT. These results suggest that TPx-1 does not prevent parasite DNA oxidation, in contrast to mammalian Prx, and that it is not essential for asexual parasite growth in mouse erythrocytes. However, Prx KO produced up to 60% fewer gametocytes, sexual-stage parasites involved in the transition between the mammalian host and the mosquito, than WT did. The peak of gametocytemia was also delayed; however, the male/female ratio of gametocytes and the exflagellation activity of male gametocytes were normal. These results suggest that TPx-1 is required for normal gametocyte development but does not affect the male/female gametocyte ratio or male gametogenesis. Although the mechanism by which PbTPx-1 contributes to gametocyte development remains unknown, these findings suggest, for the first time, the involvement of Prx in the sexual development of the malaria parasite.     

Plasmodium knowlesi: persistence of transmission blocking immunity in monkeys immunized with gamete antigens.

Eight rhesus monkeys immunized with a partially purified preparation of Plasmodium knowlesi gametes were monitored for over 6 years to determine the extent of transmission blocking immunity. Monkeys were challenged regularly, and anti-gamete antibodies were assayed by in vivo and in vitro mosquito feedings. Transmission blocking immunity persisted at high levels in most of the monkeys. In those animals in which protection waned between challenges, a challenge infection provided a sufficient booster effect to prevent infection of mosquitoes. Immunity to other stages of malaria (i.e., sporozoites and asexual erythrocyte forms) failed to induce immunity against gametes.     

Plasmodium falciparum: epidemiology and man-mosquito transmission and infection in the vector

The level of malaria transmission is usually estimated by some entomological parameters (entomological inoculation rate or reproductive rate and its seasonal variations). However, only one aspect of the malaria transmission is explored by this way, i.e. the transmission of Plasmodium from mosquito to man. The transmission from man to mosquito, the development of parasite in the mosquito midgut, and the role of transmission blocking immunity remain poorly documented. Recent studies on vaccination with gamete antigens showed that transmission blocking immunity, and the natural infectiousness of gametocytes after treatment underlined the need for taking into account a new aspect of malaria epidemiology concerning the transmission of Plasmodium from man to mosquito. In this paper, authors propose and discuss different new indicators and methods to improve our knowledge on malaria epidemiology.     

Stage-specific gametocytocidal effect in vitro of the antimalaria drug qinghaosu onPlasmodium falciparum

Qinghaosu, an anti-malaria drug, has been found to kill not only asexual blood stages but also the early stages of gametocytes ofPlasmodium falciparum. The effect of qinghaosu in vitro depends on the concentration of the drug as well as on the initial parasitemia level (IC₅₀=10–20 nM with 1% initial parasitemia). Resistance ofP. falciparum to other anti-malaria drugs, e.g., chloroquine and pyrimethamine, did not affect susceptibility of its asexual and sexual stages to qinghaosu. Gametocytocidal effect of qinghaosu may play a role in the interruption of malaria transmission.     

A model for sequestration of the transmission stages of Plasmodium falciparum: adhesion of gametocyte-infected erythrocytes to human bone marrow cells

With the aim of developing an appropriate in vitro model of the sequestration of developing Plasmodium falciparum sexual-stage parasites, we have investigated the cytoadherence of gametocytes to human bone marrow cells of stromal and endothelial origin. Developing stage III and IV gametocytes, but not mature stage V gametocytes, adhere to bone marrow cells in significantly higher densities than do asexual-stage parasites, although these adhesion densities are severalfold lower than those encountered in classical CD36-dependent assays of P. falciparum cytoadherence. This implies that developing gametocytes undergo a transition from high-avidity, CD36-mediated adhesion during stages I and II to a lower-avidity adhesion during stages III and IV. We show that this adhesion is CD36 independent, fixation sensitive, stimulated by tumor necrosis factor alpha, and dependent on divalent cations and serum components. These data suggest that gametocytes and asexual parasites utilize distinct sets of receptors for adhesion during development in their respective sequestered niches. To identify receptors for gametocyte-specific adhesion of infected erythrocytes to bone marrow cells, we tested a large panel of antibodies for the ability to inhibit cytoadherence. Our results implicate ICAM-1, CD49c, CD166, and CD164 as candidate bone marrow cell receptors for gametocyte adhesion.