Plasmodium gallinaceum preferentially invades vesicular ATPase-expressing cells in Aedes aegypti midgut

Penetration of the mosquito midgut epithelium is obligatory for the further development of Plasmodium parasites. Therefore, blocking the parasite from invading the midgut wall disrupts the transmission of malaria. Despite such a pivotal role in malaria transmission, the cellular and molecular interactions that occur during the invasion are not understood. Here, we demonstrate that the ookinetes of Plasmodium gallinaceum, which is related closely to the human malaria parasite Plasmodium falciparum, selectively invade a cell type in the Aedes aegypti midgut. These cells, unlike the majority of the cells in the midgut, do not stain with a basophilic dye (toluidine blue) and are less osmiophilic. In addition, they contain minimal endoplasmic reticulum, lack secretory granules, and have few microvilli. Instead, these cells are highly vacuolated and express large amounts of vesicular ATPase. The enzyme is associated with the apical plasma membrane, cytoplasmic vesicles, and tubular extensions of the basal membrane of the invaded cells. The high cost of insecticide use in endemic areas and the emergence of drug resistant malaria parasites call for alternative approaches such as modifying the mosquito to block the transmission of malaria. One of the targets for such modification is the parasite receptor on midgut cells. A step toward the identification of this receptor is the realization that malaria parasites invade a special cell type in the mosquito midgut.     

Rainfall, abundance of Aedes aegypti and dengue infection in Selangor, Malaysia

An epidemio-meteorotropic analytical study of Selangor, in the Southwest coast of Peninsular Malaysia, examines the monthly incidence of dengue for the period 1973-1982 to assess possible quantitative association with the monthly rainfall. The relationships between rainfall, abundance of A. aegypti and dengue infection during 1982 in Jinjang, a dengue-prone area in Selangor, were also examined. A quantitative association between rainfall and the number of dengue cases was found during the first wet period. The lag time between the onset of heavy rain and dengue outbreak was about two to three months. A 120% increase in the number of dengue cases was observed when the monthly rainfall was 300 mm or more. Positive associations were seen between the incidence of dengue and the Aedes house index and the Breteau index in Jinjang. The relationships between these three variables and rainfall suggest that the latter might have exerted its effect on dengue infection partly through the creation of more breeding sites for A. aegypti. Assessment of the importance of A. aegypti in the transmission of dengue in this locality was not possible because of the lack of adjustment for A. albopictus, the other known vector of dengue in the state, and for social and other environmental factors influencing infection rates. In spite of this and the interpretational problems common in aggregate studies, the present analyses have provided relatively strong statistical evidence of an association between rainfall and dengue outbreaks in Selangor, thereby indicating that it is a factor worthy of careful surveillance and monitoring.     

Vectored antibody gene delivery protects against Plasmodium falciparum sporozoite challenge in mice

Malaria caused by Plasmodium falciparum kills nearly one million children each year and imposes crippling economic burdens on families and nations worldwide. No licensed vaccine exists, but infection can be prevented by antibodies against the circumsporozoite protein (CSP), the major surface protein of sporozoites, the form of the parasite injected by mosquitoes. We have used vectored immunoprophylaxis (VIP), an adeno-associated virus-based technology, to introduce preformed antibody genes encoding anti-P. falciparum CSP mAb into mice. VIP vector-transduced mice exhibited long-lived mAb expression at up to 1,200 µg/mL in serum, and up to 70% were protected from both i.v. and mosquito bite challenge with transgenic Plasmodium berghei rodent sporozoites that incorporate the P. falciparum target of the mAb in their CSP. Serum antibody levels and protection from mosquito bite challenge were dependent on the dose of the VIP vector. All individual mice expressing CSP-specific mAb 2A10 at 1 mg/mL or more were completely protected, suggesting that in this model system, exceeding that threshold results in consistent sterile protection. Our results demonstrate the potential of VIP as a path toward the elusive goal of immunization against malaria.Among infectious diseases, malaria ranks fourth as a cause of death. In Africa in 2012, 500,000–800,000 deaths, mostly among children under 5 y of age, resulted from ∼200 million clinical cases of malaria caused by Plasmodium falciparum, the parasite species responsible for most malaria mortality (1). In addition to its direct effect on health, malaria imposes severe economic burdens. These include crippling treatment costs at the level of individual families and a significant contribution to low national incomes and reduced overall rates of economic growth in nations worldwide (2, 3). The human and economic burdens imposed by malaria make malaria reduction a critical global priority.Traditional approaches to malaria control such as antimalarial drug treatment, mosquito control by habitat modification and insecticide use, and reduction of exposure to infected mosquitoes have had substantial success in reducing malaria incidence and malaria-specific mortality rates (4). However, these achievements may be difficult to sustain in the face of growing drug resistance among parasites, insecticide resistance in vector populations, and the reduced funding for malaria control anticipated by the World Health Organization for the near future (4). If continued progress against malaria is to be made, it is essential that new approaches for malaria prevention be added to currently available tools.Sporozoites are the infectious form of the malaria parasite injected by Anopheles mosquitoes. It has been known for decades that immunization of animals or humans with radiation-attenuated sporozoites can elicit sterilizing immunity to malaria, preventing infection, pathogenesis, and transmission (5–8). The predominant antibody response to immunization by irradiated sporozoites is to the circumsporozoite protein (CSP), which coats the sporozoite surface (9, 10). In in vivo and in vitro models of P. falciparum sporozoite infection, infection can be blocked completely by antibody to the immunodominant epitope of CSP, a tetrapeptide [asn-ala-asn-pro (NANP)] found in 30 or more tandemly repeated copies in the central region of the protein (11–13). The NANP repeat is stringently conserved in P. falciparum isolates from diverse geographical locations (14) and thus represents a potentially universal target for P. falciparum immunity. The most advanced malaria infection-blocking vaccine candidate, RTS,S, is comprised of hepatitis B virus-like particles that display a carboxyl-terminal segment of P. falciparum CSP, including part of the central NANP repeat region. Immunization with RTS,S reduces incidence of clinical malaria by 40–70% in children, but levels of protection wane with time (15–18).Vectored immunoprophylaxis (VIP) provides an alternative to conventional immunization as a route to protective antibody expression (19–21). VIP employs optimized adeno-associated virus (AAV) based vectors to deliver genes encoding mAb with previously characterized specificities to animals. Intramuscular injection of VIP vectors in mice and macaques elicits long-lived antibody or antibody-related immunoadhesin production at levels sufficient to protect against HIV, simian immunodeficiency virus, and influenza A virus infection (19–22). Here we report that in mice, VIP-directed production of sporozoite-neutralizing mAb against the P. falciparum CSP central repeat can confer sterile immunity to infection by a transgenic strain of the rodent parasite Plasmodium berghei whose CSP contains the P. falciparum CSP central repeat [Pb/Pf (12)].     

Safety and immunogenicity of a recombinant sporozoite malaria vaccine against Plasmodium vivax.

A recombinant Plasmodium vivax circumsporozoite (CS) antigen representing approximately 70% of the CS protein was expressed in yeast and adsorbed onto aluminum hydroxide for use as a malaria vaccine. In a study of safety and immunogenicity, 30 volunteers were divided into four groups of 5, 5, 10, and 10 individuals, and inoculated intramuscularly with 50, 100, 200, or 400 micrograms of vaccine, respectively. Primary vaccinations were followed by two booster immunizations at six weeks and six months. Overall, the vaccine was well tolerated. Following the third vaccination, one volunteer developed acute hepatitis of uncertain etiology that resolved without sequelae. All volunteers in the 400-micrograms group, and six of 10 in the 200-micrograms group generated IgG against P. vivax CS protein, as determined by Western blot using recombinant CS protein. However, the magnitude of the antibody response measured by indirect immunofluorescence of intact sporozoites or enzyme-linked immunosorbent assay against the recombinant protein was low, and responses could not be boosted. Antigen-driven replication studies using peripheral blood lymphocytes failed to detect proliferative responses specific to peptide sequences represented in the recombinant vaccine, except in one volunteer. Minimal humoral and cell-mediated immune responses developed in most recipients who received this recombinant CS vaccine.     

Pattern of relapses in sporozoite induced Plasmodium cynomolgi B infection in rhesus monkeys

The pattern of sequential relapses in 10 rhesus monkeys following inoculation of sporozoites of Plasmodium cynomolgi B has been studied after administering curative dose of chloroquine (5 mg/kg base X 7 days) to eliminate blood parasitaemia after each relapse. Observation for periods ranging from 109 to 245 days showed that the interval between first six relapses was 19.3 +/- 6.77 days (1st relapse), 20.9 +/- 8.43 days (2nd relapse), 22.8 +/- 8.55 days (3rd relapse), 27.8 +/- 10.0 days (4th relapse), 31.67 +/- 11.50 days (5th relapse) and 32.5 +/- 16.26 days (6th relapse). The results of this study indicate a gradual extension of the relapse interval in successive relapses.     

Association of human immune response to Aedes aegypti salivary proteins with dengue disease severity

Dengue viruses (DENV; family Flaviviridae, genus Flavivirus) are transmitted by Aedes aegypti mosquitoes and can cause dengue fever (DF), a relatively benign disease, or more severe dengue haemorrhagic fever (DHF). Arthropod saliva contains proteins delivered into the bite wound that can modulate the host haemostatic and immune responses to facilitate the intake of a blood meal. The potential effects on DENV infection of previous exposure to Ae. aegypti salivary proteins have not been investigated. We collected Ae. aegypti saliva, concentrated the proteins and fractionated them by nondenaturing polyacrylamide gel electrophoresis (PAGE). By the use of immunoblots, we analysed reactivity with the mosquito salivary proteins (MSP) of sera from 96 Thai children diagnosed with secondary DENV infections leading either to DF or DHF, or with no DENV infection, and found that different proportions of each patient group had serum antibodies reactive to specific Ae. aegypti salivary proteins. Our results suggest that prior exposure to MSP might play a role in the outcome of DENV infection in humans.     

Inability of Plasmodium knowlesi sporozoites to invade Anopheles freeborni salivary glands

Oocysts of Plasmodium knowlesi developed normally on the gut of the mosquito Anopheles freeborni, rupturing and releasing sporozoites at 10-14 days post-infection. Subsequently, however, sporozoites were never found in this mosquito's salivary glands. Heterologous transplants of whole salivary glands from uninfected An. freeborni and An. dirus, a completely susceptible mosquito, into the abdomens of insects heavily infected with mature oocysts were done. Sporozoites failed to infect An. freeborni glands implanted in An. dirus but did enter An. dirus glands implanted in An. freeborni. These experiments suggest that P. knowlesi sporozoites are unable to recognize An. freeborni glands.     

Mosquito infestation and dengue virus infection in Aedes aegypti females in schools in Merida, Mexico

We determined abundance of Aedes aegypti mosquitoes and presence of dengue virus (DENV) in females collected from schools in Mérida, México, during 2008 and 2009. Backpack aspiration from 24 schools produced 468 females of Ae. aegypti and 1,676 females of another human biter, Culex quinquefasciatus. Ae. aegypti females were collected most commonly from classrooms followed by offices and bathrooms. Of these females, 24.7% were freshly fed. Examination of 118 pools of Ae. aegypti females (total of 415 females) for presence of DENV RNA produced 19 positive pools (16.1%). DENV-infected pools were detected from 11 (45.8%) of 24 schools and came from different room types, including classrooms, offices, and bathrooms. The overall rate of DENV infection per 100 Ae. aegypti females was 4.8. We conclude that schools in Mérida present a risk environment for students, teachers, and other personnel to be exposed to mosquitoes and bites of DENV-infected Ae. aegypti females.     

Seasonal prevalence of Aedes aegypti immatures in Kolkata, India

Dengue and dengue hemorrhagic fever (DHF) is recurring in India in recent years, affecting many cities including Kolkata (Calcutta). In view of this, a survey of three years was carried out on the prevalence of immatures of the vector mosquito Aedes aegypti in the city of Kolkata. Mosquito immatures were found throughout the year with fluctuating densities in the habitats surveyed. An index of density of immatures (I/S)m employed in the study revealed that in the months of August and September a peak in abundance occurs, irrespective of the habitats, with the values ranging between 1.12 and 11.4. The (I/S)m values were low during the months of April and May ranging between 0.4 and 3.8. As larval habitats, earthen containers and the sewage drains were positive throughout the year, while during the months of December to February, air-coolers and conditioners as habitats remained totally dry, without immature Ae. aegypti. The number of positive sites and monthly density between the habitat types were significantly different with an I/S- value (for a year) ranging between 2.64 (air-coolers) and 4.97 (earthen containers). The survey, while providing preliminary baseline data on immature abundance in Kolkata, calls for continuous entomological surveillance using standard protocols as carried out in Thailand for the vector management program against dengue.     

Dengue 3 virus infection of Aedes albopictus and Aedes aegypti: comparison of parent and progeny candidate vaccine viruses

DEN-3 parent (strain CH53489) and progeny candidate vaccine (clone 24/28) viruses were compared in their abilities to interact with Aedes aegypti and Ae. albopictus. The parent and progeny virus were equivalent in their ability to infect, to replicate in, and to be transmitted by both species of mosquitoes. The candidate vaccine DEN-3 clone was temperature sensitive and had small plaque morphology. These phenotypic markers remained stable during mosquito passage. Thus, temperature sensitivity and small plaque morphology are not reliable biological markers for attenuation.     

In vitro damage of cultured ookinetes of Plasmodium gallinaceum by digestive proteinases from susceptible Aedes aegypti.

After exposure to extracts from blood fed A. aegupti cultured ookinetes of P. gallinaceum were damaged to various, defined extents. Immature ookinetes were found to be more sensitive to damage than mature ones. The damage was dependent on the digestion time after which the Aedes extracts had been prepared and could be correlated with the proteolytic activity in the extracts. Control experiments demonstrated that the factors responsible for damage were neither present in unfed mosquitoes nor in blood alone and that the damage was not a result of osmotic stress. After the treatment of the Aedes extracts with lima bean trypsin inhibitor the ookinete damage was much less, indicating that the Aedes trypsin was the major agent of damage. These results were supported by experiments in which the tryptic activity of the extracts was eliminated by thermal denaturation. It is concluded that in the mosquito midgut most of the ookinetes are damaged by digestive enzymes and that this is one factor leading to the discrepancy between the number of ingested macrogametocytes and the number of oocysts which is usually found in nature. It seems that the only ookinetes which have a chance of surviving are those which develop in the centre of the blood clot, away from the site of enzyme action.