Flying vaccinator; a transgenic mosquito delivers a Leishmania vaccine via blood feeding

‘Flying vaccinator’ is the concept of using genetically engineered hematophagous insects to deliver vaccines. Here we show the generation of a transgenic anopheline mosquito that expresses the Leishmania vaccine candidate, SP15, fused to monomeric red fluorescent protein (mDsRed) in its salivary glands. Importantly, mice bitten repeatedly by the transgenic mosquitoes raised anti‐SP15 antibodies, indicating delivery of SP15 via blood feeding with its immunogenicity intact. Thus, this technology makes possible the generation of transgenic mosquitoes that match the original concept of a ‘flying vaccinator’. However, medical safety issues and concerns about informed consent mitigate the use of the ‘flying vaccinator’ as a method to deliver vaccines. We propose that this expression system could be applied to elucidate saliva–malaria sporozoite interactions.     

Visualization and live imaging analysis of a mosquito saliva protein in host animal skin using a transgenic mosquito with a secreted luciferase reporter system

Mosquitoes inject saliva into a vertebrate host during blood feeding. The analysis of mosquito saliva in host skin is important for the elucidation of the inflammatory responses to mosquito bites, the development of antithrombotic drugs, and the transmission‐blocking of vector‐borne diseases. We produced transgenic Anopheles stephensi mosquitoes expressing the secretory luciferase protein (MetLuc) fused to a saliva protein (AAPP) in the salivary glands. The transgene product (AAPP‐MetLuc) of transgenic mosquitoes exhibited both luciferase activity as a MetLuc and binding activity to collagen as an AAPP. The detection of luminescence in the skin of mice bitten by transgenic mosquitoes showed that AAPP‐MetLuc was injected into the skin as a component of saliva via blood feeding. AAPP‐MetLuc remained at the mosquito bite site in host skin with luciferase activity for at least 4 h after blood feeding. AAPP was also suspected of remaining at the site of injury caused by the mosquito bite and blocking platelet aggregation by binding to collagen. These results demonstrated the establishment of visualization and time‐lapse analysis of mosquito saliva in living vertebrate host skin. This technique may facilitate the analysis of mosquito saliva after its injection into host skin, and the development of new drugs and disease control strategies.     

Induction of antisporozoite antibodies by biting of transgenic Anopheles stephensi delivering malarial antigen via blood feeding

We produced a transgenic mosquito expressing a rodent malaria vaccine candidate antigen in the salivary gland. Three tandemly repeated amino acid units from the repeat region of circumsporozoite protein of Plasmodium berghei (PbCS3R) fused to red fluorescent protein (monomeric DsRed) was chosen as a vaccine candidate antigen. Immunoblot and fluorescence microscopic analyses showed the transgene expression in the female salivary gland. The transgene product was released from the proboscis as a component of saliva. The monomeric DsRed-fusion expression system could be suitable for transgene secretion in the saliva of female mosquitoes. Mice repeatedly bitten by transgenic mosquitoes raised antibodies against P. berghei sporozoites, and the sera had protective ability against sporozoite invasion of human hepatoma HepG2 cells. These results suggest that transgene products are immunogenically active in saliva, and induce the antibodies to malaria parasite. These findings indicate that this technology has the potential for production of a 'flying vaccinator' for rodent malaria parasites.     

Developmentally regulated infectivity of malaria sporozoites for mosquito salivary glands and the vertebrate host

Sporozoites are an invasive stage of the malaria parasite in both the mosquito vector and the vertebrate host. We developed an in vivo assay for mosquito salivary gland invasion by preparing Plasmodium gallinaceum sporozoites from infected Aedes aegypti mosquitoes under physiological conditions and inoculating them into uninfected female Ae. aegypti. Sporozoites from mature oocysts were isolated from mosquito abdomens 10 or 11 d after an infective blood meal. Salivary gland sporozoites were isolated 13 or 14 d after an infective blood meal. Purified oocyst sporozoites that were inoculated into uninfected female mosquitoes invaded their salivary glands. Using the same assay system, sporozoites derived from salivary glands did not reinvade the salivary glands after inoculation. Conversely, as few as 10 to 50 salivary gland sporozoites induced infection in chickens, while only 2 of 10 chickens inoculated with 5,000 oocyst sporozoites were infected. Both sporozoite populations were found to express a circumsporozoite protein on the sporozoite surface as determined by immunofluorescence assay and circumsporozoite precipitation test using a circumsporozoite protein-specific monoclonal antibody. We conclude that molecules other than this circumsporozoite protein may be responsible for the differential invasion of mosquito salivary glands or infection of the vertebrate host.     

A Class of Tricyclic Compounds Blocking Malaria Parasite Oocyst Development and Transmission

Malaria is a deadly infectious disease in many tropical and subtropical countries. Previous efforts to eradicate malaria have failed, largely due to the emergence of drug-resistant parasites, insecticide-resistant mosquitoes and, in particular, the lack of drugs or vaccines to block parasite transmission. ATP-binding cassette (ABC) transporters are known to play a role in drug transport, metabolism, and resistance in many organisms, including malaria parasites. To investigate whether a Plasmodium falciparum ABC transporter (Pf14_0244 or PfABCG2) modulates parasite susceptibility to chemical compounds or plays a role in drug resistance, we disrupted the gene encoding PfABCG2, screened the recombinant and the wild-type 3D7 parasites against a library containing 2,816 drugs approved for human or animal use, and identified an antihistamine (ketotifen) that became less active against the PfABCG2-disrupted parasite in culture. In addition to some activity against asexual stages and gametocytes, ketotifen was highly potent in blocking oocyst development of P. falciparum and the rodent parasite Plasmodium yoelii in mosquitoes. Tests of structurally related tricyclic compounds identified additional compounds with similar activities in inhibiting transmission. Additionally, ketotifen appeared to have some activity against relapse of Plasmodium cynomolgi infection in rhesus monkeys. Further clinical evaluation of ketotifen and related compounds, including synthetic new derivatives, in blocking malaria transmission may provide new weapons for the current effort of malaria eradication.     

Two-dimensional gel analysis of midgut proteins of Anopheles stephensi lines with different susceptibility to Plasmodium falciparum infection

Little is known about the composition of the mosquito midgut which plays a central role in the development and subsequent transmission of malaria parasites. As a first step towards the characterization of mosquito midgut molecules involved in the transmission of malaria parasites, we analysed two-dimensional gel electrophoresis patterns of the midgut proteins of sugar-fed and blood-fed Anopheles stephensi lines of different susceptibility to P. falciparum infection. Two lines fully susceptible and one line (Pb3-9A) of reduced susceptibility were used. In the refractory line ookinetes do develop but are only inefficiently transformed into oocysts (Feldmann & Ponnudurai, 1989). The protein profiles of midguts from all sugar-fed mosquito lines were similar. However, after blood feeding, the midgut of the fully susceptible lines contained proteins not found in the midgut of line Pb3-9A. Twenty-nine such proteins were detected and are candidates for involvement in the interaction between the mosquito midgut and P. falciparum.     

Robust salivary gland-specific transgene expression in Anopheles stephensi mosquito

Malaria sporozoites invade the mosquito salivary glands and wait in the salivary duct until the next blood feeding. The mechanisms of the process and molecules involved in the salivary gland invasion remain largely unknown. To establish a robust salivary gland-specific transgene expression in Anopheles stephensi, we obtained a salivary gland-specific promoter for a gene encoding anopheline antiplatelet protein (AAPP). The aapp promoter is a female salivary gland-specific and blood meal-inducible strong promoter. Using this promoter, we generated a transgenic An. stephensi expressing abundant Discosoma sp. red fluorescent protein (DsRed) in the distal-lateral lobes of the glands, where the sporozoites invade preferentially. These results open up the possibilities of elucidating salivary gland-parasite interactions and generating transgenic mosquitoes refractory to parasites.     

Knockdown of mitogen‐activated protein kinase (MAPK) signalling in the midgut of Anopheles stephensi mosquitoes using antisense morpholinos

Arthropod‐borne infectious diseases are responsible for nearly 1.5 million deaths annually across the globe, with malaria responsible for >50% of these deaths. Recent efforts to enhance malaria control have focused on developing genetically modified Anopheles mosquitoes that are resistant to malaria parasite infection by manipulating proteins that are essential to the immune response. Although this approach has shown promise, the lack of efficient genetic tools in the mosquito makes it difficult to investigate innate immunity using reverse genetics. Current gene knockdown strategies based on small interfering RNA are typically labourious, inefficient, and require extensive training. In the present study, we describe the use of morpholino antisense oligomers to knockdown MEK‐ERK signalling in the midgut of Anopheles stephensi through a simple feeding protocol. Anti‐MEK morpholino provided in a saline meal was readily ingested by female mosquitoes with minimal toxicity and resulted in knockdown of total MEK protein levels 3–4 days after morpholino feeding. Further, anti‐MEK morpholino feeding attenuated inducible phosphorylation of the downstream kinase ERK and, as predicted by previous work, reduced parasite burden in mosquitoes infected with Plasmodium falciparum. To our knowledge, this is the first example of morpholino use for target protein knockdown via feeding in an insect vector. Our results suggest this method is not only efficient for studies of individual proteins, but also for studies of phenotypic control by complex cell signalling networks. As such, our protocol is an effective alternative to current methods for gene knockdown in arthropods.     

Comparative fitness assessment of Anopheles stephensi transgenic lines receptive to site‐specific integration

Genetically modified mosquitoes that are unable to transmit pathogens offer opportunities for controlling vector‐borne diseases such as malaria and dengue. Site‐specific gene recombination technologies are advantageous in the development of these insects because antipathogen effector genes can be inserted at integration sites in the genome that cause the least alteration in mosquito fitness. Here we describe Anopheles stephensi transgenic lines containing ?C31 attP‘docking’ sites linked to a fluorescent marker gene. Chromosomal insertion sites were determined and life‐table parameters were assessed for transgenic mosquitoes of each line. No significant differences in fitness between the transgenic and nontransgenic mosquitoes were detected in this study. These transgenic lines are suitable for future site‐specific integrations of antiparasite transgenes into the attP sites.     

Therapeutic plasma exchange in the treatment of complicated Plasmodium falciparum malaria: A case report

Severe falciparum malaria is associated with multiple organ dysfunction and a high rate of fatal outcome. Malaria is a world‐wide disease in tropical areas through the bites of vector mosquitoes. Parasitic protozoans introduced by the mosquito's saliva to the blood travel to the liver then mature and reproduce. In humans, malaria is caused by Plasmodium falciparum, P. malariae, P. ovale, P. vivax, and P. knowlesi, and P. falciparum causes most deaths. Typical malaria symptoms include fever, chills, fatigue, headache, nausea, and vomiting. In severe cases, it can cause jaundice, seizures, coma, or death. Jaundice, caused by intravascular hemolysis is a usual complication of malaria, especially in patients with P. falciparum infection. The use of exchange transfusion in malaria is not currently advocated by the Centers of Disease Control and Prevention (CDC) of the United States of America. The role of therapeutic plasma exchange as an adjunctive therapy in malaria has not been widely discussed in the literature. Here, we present a 23‐year‐old patient with jaundice, acute renal failure, and cerebral involvement who was successfully treated with plasma exchange and hemodialysis.     

Survival of Plasmodium falciparum in human blood during refrigeration

BACKGROUND: Transfusion‐transmitted malaria remains a serious concern for blood safety. Viable Plasmodium parasites must be present in human blood to transmit malaria, but their survival in blood over time stored under refrigeration has never been carefully investigated. STUDY DESIGN AND METHODS: We spiked leukoreduced normal human blood with Plasmodium falciparum (3D7 strain) asexual ring‐stage parasites and stored it at 4°C for 28 days, taking samples at different days intervals. We evaluated the samples for parasitemia by blood film microscopy and by culturing red blood cells (RBCs) to allow further development of parasites. RESULTS: We observed a significant reduction in parasitemia (0.5% vs. 0.12%) after only 1 day in storage at 4°C. Thereafter, reduction in parasitemia was relatively gradual. Microscopically detectable parasites were present even after 28 days of storage. However, after storing for more than 14 days at 4°C, parasites no longer replicated when cultured in vitro. CONCLUSION: Although the storage of asexual blood‐stage P. falciparum parasites at 4°C is detrimental to their survival (a 7.1‐fold reduction in parasitemia after 14 days in storage), parasites remained microscopically detectable for 28 days, the end time point of our study. Further in vitro and in vivo studies will be needed to confirm loss of viability of P. falciparum after 14 days in storage, but our initial efforts repeatedly failed to show maturation and development of the parasites in cultured RBCs after that time.     

Insecticide resistance in mosquitoes and failure of malaria control

Evaluation of: Corbel V, Akogbeto M, Damien GB et al. Combination of malaria vector control interventions in pyrethroid resistance area in Benin: a randomized cluster controlled trial. Lancet Infect. Dis. 12(8), 617–626 (2012).

Major efforts are being made to achieve the goals set by the World Health Assembly and the Roll Back Malaria partnership for malaria control. The paper under evaluation demonstrates that these efforts should be evaluated regularly as sometimes multifaceted interventions are not more effective that one single targeted intervention which is costless. Moreover, the increasing antimalarial drug resistance of Plasmodium falciparum to artemisinin, the almost complete resistance of mosquitoes to pyrethroid, the likely zoonotic nature of P. falciparum and the behavioral changes of mosquitoes are many new features that indicate that malaria control is not yet achieved.     

Circumsporozoite protein as a potential target for antimalarials

Since the discovery of circumsporozoite protein (CSP), a major sporozoite surface antigen, by Ruth Nussenzweig and Victor Nussenzweig in the early 1980s, the role of CSP in protection against malaria has been extensively investigated. Several monoclonal antibodies against CSP have been generated to date, with some of them mediating antimalarial protection upon passive transfer into animals. Genetically engineered transgenic mosquitoes producing the anti-CSP antibody have recently been generated to reduce malarial transmission. A monoclonal anti-CSP antibody was produced in mice by adeno-associated virus vector, which protected them from malaria. Phase III trials with RTS,S vaccine that targets CSP of Plasmodium falciparum have shown modest efficacy. Polyclonal anti-CSP antibodies derived from children who received the RTS,S vaccine failed to block malarial transmission through mosquitoes, but passive transfer of monoclonal antibodies raised from RTS,S-vaccinated recipient conferred protection against malaria in mice. Taken together, these findings may imply CSP as an antimalarial target.     

4-(1H)-Quinolones and 1,2,3,4-Tetrahydroacridin-9(10H)-Ones Prevent the Transmission of Plasmodium falciparum to Anopheles freeborni

Malaria kills approximately 1 million people a year, mainly in sub-Saharan Africa. Essential steps in the life cycle of the parasite are the development of gametocytes, as well as the formation of oocysts and sporozoites, in the Anopheles mosquito vector. Preventing transmission of malaria through the mosquito is necessary for the control of the disease; nevertheless, the vast majority of drugs in use act primarily against the blood stages. The study described herein focuses on the assessment of the transmission-blocking activities of potent antierythrocytic stage agents derived from the 4(1H)-quinolone scaffold. In particular, three 3-alkyl- or 3-phenyl-4(1H)-quinolones (P4Qs), one 7-(2-phenoxyethoxy)-4(1H)-quinolone (PEQ), and one 1,2,3,4-tetrahydroacridin-9(10H)-one (THA) were assessed for their transmission-blocking activity against the mosquito stages of the human malaria parasite (Plasmodium falciparum) and the rodent parasite (P. berghei). Results showed that all of the experimental compounds reduced or prevented the exflagellation of male gametocytes and, more importantly, prevented parasite transmission to the mosquito vector. Additionally, treatment with ICI 56,780 reduced the number of sporozoites that reached the Anopheles salivary glands. These findings suggest that 4(1H)-quinolones, which have activity against the blood stages, can also prevent the transmission of Plasmodium to the mosquito and, hence, are potentially important drug candidates to eradicate malaria.     

MAEBL is essential for malarial sporozoite infection of the mosquito salivary gland

Malarial sporozoites mature in the oocysts formed in the mosquito midgut wall and then selectively invade the salivary glands, where they wait to be transmitted to the vertebrate host via mosquito bite. Invasion into the salivary gland has been thought to be mediated by specific ligand-receptor interactions, but the molecules involved in these interactions remain unknown. MAEBL is a single transmembrane-like protein that is structurally related to merozoite adhesive proteins. We found MAEBL of the rodent malaria parasite, Plasmodium berghei, to be specifically produced by the sporozoites in the oocyst and localized in their micronemes, which are secretory organelles involved in malarial parasite invasion into the host cell. A targeted disruption experiment of the P. berghei MAEBL gene revealed that it was essential for sporozoite infection of the salivary gland and was involved in the attachment to the salivary gland surface. In contrast, the disruption of the MAEBL gene did not affect sporozoite motility in vitro nor infectivity to the vertebrate host. These results suggest that P. berghei MAEBL is a sporozoite attachment protein that participates in specific binding to and infection of the mosquito salivary gland.     

Childhood Plasmodium falciparum malaria complicated by splenic abscess

We report on an 8‐year‐old girl with Plasmodium falciparum malaria complicated by splenic abscess during the course of her infection. The diagnosis of malaria was based upon the demonstration of trophozoites of P. falciparum in the peripheral blood smear. The trophozoites of the same organism were visualized on the Giemsa‐stained smear prepared from the percutaneous splenic aspirate specimen. The patient failed to respond to parenteral Artesunate alone, which demanded emergent splenectomy. To our knowledge, this is the first report of splenic abscess complicating the course of childhood P. falciparum malaria in the English literature.     

Asymptomatic carriage of malaria parasites in blood donors in Yaoundé

Background: Malaria can be transmitted through blood transfusion, but there is paucity of data concerning transfusion‐transmitted malaria in Cameroun. Objective: To determine prevalence of malaria infection and association with epidemiological and clinical data obtained from donors' responses. Methods: Microscopic examination of stained thick and thin blood smears for the detection, quantification and specification of Plasmodium sp was performed on 493 blood donors in two main hospitals in Yaoundé during October and November 2007. Results: Overall 6·5% of blood donors were detected positive for Plasmodium sp infection: 90·6% was Plasmodium falciparum and 9·4% was Plasmodium malariae. Parasite counts ranged from 80 to 800 µL^?1 with a median of 320 µL^?1. Asexual and sexual forms were found in 75·9 and 24·1% of cases, respectively. Age, sex, type of blood donor (voluntary non‐remunerated vs familial/replacement) and fate of blood donation (selected vs discarded) did not affect the prevalence of malaria carriage. The lack of malaria prophylaxis as well as the manifestation of malaria symptoms within 2 weeks and 1 month preceding blood donation were significantly associated with high frequency of parasites carriage. Conclusion: Malaria parasites carriage is frequent among blood donors in Yaoundé. These data seem to describe high‐risk donor profile and may help improving blood safety related to transfusion‐transmitted malaria in Cameroon.