Plasmodium Transmission Research Articles

A semi-automated luminescence based standard membrane feeding assay identifies novel small molecules that inhibit transmission of malaria parasites by mosquitoes.

Current first-line treatments for uncomplicated falciparum malaria rapidly clear the asexual stages of the parasite, but do not fully prevent parasite transmission by mosquitoes. The standard membrane feeding assay (SMFA) is the biological gold standard assessment of transmission reducing activity (TRA), but its throughput is limited by the need to determine mosquito infection status by dissection and microscopy. Here we present a novel dissection-free luminescence based SMFA format using a transgenic Plasmodium falciparum reporter parasite without resistance to known antimalarials and therefore unrestricted in its utility in compound screening. Analyses of sixty-five compounds from the Medicines for Malaria Venture validation and malaria boxes identified 37 compounds with high levels of TRA (>80%); different assay modes allowed discrimination between gametocytocidal and downstream modes of action. Comparison of SMFA data to published assay formats for predicting parasite infectivity indicated that individual in vitro screens show substantial numbers of false negatives. These results highlight the importance of the SMFA in the screening pipeline for transmission reducing compounds and present a rapid and objective method. In addition we present sixteen diverse chemical scaffolds from the malaria box that may serve as a starting point for further discovery and development of malaria transmission blocking drugs.

Investigation on possible transmission of monkeys' Plasmodium to human in a populations living in the equatorial rainforest of the Democratic republic of Congo.

Plasmodiums are protozoa that may infect various hosts. Only five species are now recognized as naturally parasitizing humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. This fifth species, P. knowlesi, previously identified as naturally parasitizing the monkey Macaca fascicularis, has been microscopically confused for a long time with P. malariae or P. falciparum and it was not possible to correctly differentiate them until the advent of molecular biology. To date, natural human infections with P. knowlesi only occur in Southeast Asia and a similar phenomenon of natural transmission of simian plasmodium to humans has not been reported elsewhere. This study was conducted to investigate a possible transmission of African small monkey's plasmodium to humans in populations living near the rainforest of the Democratic Republic of Congo (DRC) where several species of non-human primates are living. Two successive real-time PCRs were identified in the literature and used in combination for purpose. Only P. falciparum was found in this study. However, studies with larger samples and with more advanced techniques should be conducted.

Avian Malaria is Absent in Juvenile Colonial Herons (Ardeidae) but notCulex pipiensMosquitoes in the Camargue, Southern France

Apicomplexan blood parasites Plasmodium and Haemoproteus (together termed “Avian malaria”) and Leucocytozoon are widespread, diverse vector-transmitted blood parasites of birds, and conditions associated with colonial nesting in herons (Ardeidae) and other waterbirds appear perfect for their transmission. Despite studies in other locations reporting high prevalence of parasites in juvenile herons, juvenile Little Egrets (Egretta garzetta) previously tested in the Camargue, Southern France, had a total absence of malaria parasites. This study tested the hypotheses that this absence was due to insufficient sensitivity of the tests of infection; an absence of infective vectors; or testing birds too early in their lives. Blood was sampled from juveniles of four species shortly before fledging: Little Egret (n = 40), Cattle Egret (Bubulcus ibis; n = 40), Black-crowned Night-Heron (Nycticorax nycticorax, n = 40), and Squacco Heron (Ardeola ralloides; n = 40). Sensitive nested-Polymerase Chain Reaction was used to test for the presence of parasites in both birds and host-seeking female mosquitoes captured around the colonies. No malaria infection was found of in any of the heron species. Four different lineages of Plasmodium were detected in pooled samples of female Culex pipiens mosquitoes, including two in potentially infective mosquitoes. These results confirm that the absence of malaria parasites previously demonstrated in Little Egret is not due to methodological limitations. Although the prevalence of infection in mosquitoes was low, conditions within the colonies were suitable for transmission of Plasmodium. These colonial heron species may have evolved strategies for resisting malaria infection through physiological or behavioral mechanisms.

Simian malaria in wild macaques: first report from Hulu Selangor district, Selangor, Malaysia.

BackgroundMalaria is a vector-borne parasitic disease which is prevalent in many developing countries. Recently, it has been found that Plasmodium knowlesi, a simian malaria parasite can be life-threatening to humans. Long-tailed macaques, which are widely distributed in Malaysia, are the natural hosts for simian malaria, including P. knowlesi. The aim of the present study was to determine the prevalence of simian malaria parasites in long-tailed macaques in the district of Hulu Selangor, Selangor, Malaysia.MethodsA total of 70 blood samples were collected from Macaca fascicularis dwelling in the forest of Hulu Selangor by the Department of Wildlife and National Parks Peninsular Malaysia, Kuala Lumpur, Malaysia. DNA was extracted using PureLink™ Genomic DNA Kits. Conventional and nested PCR were used to detect the genus and species of Plasmodium parasites respectively. In addition, phylogenetic analysis was carried out to confirm the species of Plasmodium parasites.ResultsThirty-five (50 %) of the 70 samples were positive for Plasmodium using genus-specific primers. These positive samples were then subjected to nested PCR targeting the 18S ribosomal RNA genes to detect all five simian malaria parasites: namely, P. knowlesi, Plasmodium inui, Plasmodium cynomolgi, Plasmodium fieldi, and Plasmodium coatneyi. All five species of simian malaria parasites were detected. Of these, P. inui was the predominant (65.7 %), followed by P. knowlesi (60 %), P. cynomolgi (51.4 %) P. coatneyi (45.7 %) and P.fieldi (2.9 %). A total of nine macaques had mono-infection with P. knowlesi (four), P. cynomolgi (two), P. coatneyi (two) and P. fieldi (one). Eleven of the macaques had dual infections while 12 had triple infections. Three macaques were infected with four species of Plasmodium. Molecular and phylogenetic analysis confirmed the five species of Plasmodium parasites.ConclusionThis study has provided evidence to elucidate the presence of transmission of malaria parasites among the local macaques in Hulu Selangor. Since malaria is a zoonosis, it is important to determine the new control strategies for the control of malaria.

The relationship between wing length, blood meal volume, and fecundity for seven colonies of Anopheles species housed at the Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand

Established colonies of Anopheles campestris, Anopheles cracens, Anopheles dirus, Anopheles kleini, Anopheles minimus, Anopheles sawadwongporni, and Anopheles sinensis are maintained at the Armed Forces Research Institute of Medical Sciences (AFRIMS). Females were provided blood meals on human blood containing citrate as an anticoagulant using an artificial membrane feeder. The mean wing length, used as an estimate of body size, for each species was compared to blood-feeding duration (time), blood meal volume, and numbers of eggs oviposited. Except for An. campestris and An. cracens, there were significant interspecies differences in wing length. The mean blood meal volumes (mm3) of An. kleini and An. sinensis were significantly higher than the other 5 species. For all species, the ratios of unfed females weights/blood meal volumes were similar (range: 0.76–0.88), except for An. kleini (1.08) and An. cracens (0.52), that were significantly higher and lower, respectively. Adult females were allowed to feed undisturbed for 1, 3, and 5min intervals before blood feeding was interrupted. Except for An. campestris and An. sawadwongporni, the number of eggs oviposited were significantly higher for females that fed for 3min when compared to those that only fed for 1min. This information is critical to better understand the biology of colonized Anopheles spp. and their role in the transmission of malaria parasites as they relate to the relative size of adult females, mean volumes of blood of engorged females for each of the anopheline species, and the effect of blood feeding duration on specific blood meal volumes and fecundity.

Estimating malaria transmission from humans to mosquitoes in a noisy landscape.

A basic quantitative understanding of malaria transmission requires measuring the probability a mosquito becomes infected after feeding on a human. Parasite prevalence in mosquitoes is highly age-dependent, and the unknown age-structure of fluctuating mosquito populations impedes estimation. Here, we simulate mosquito infection dynamics, where mosquito recruitment is modelled seasonally with fractional Brownian noise, and we develop methods for estimating mosquito infection rates. We find that noise introduces bias, but the magnitude of the bias depends on the ‘colour' of the noise. Some of these problems can be overcome by increasing the sampling frequency, but estimates of transmission rates (and estimated reductions in transmission) are most accurate and precise if they combine parity, oocyst rates and sporozoite rates. These studies provide a basis for evaluating the adequacy of various entomological sampling procedures for measuring malaria parasite transmission from humans to mosquitoes and for evaluating the direct transmission-blocking effects of a vaccine.

Evidence for temporal population replacement and the signature of ecological adaptation in a major Neotropical malaria vector in Amazonian Peru.

BackgroundThe major Neotropical malaria vector, Anopheles darlingi, was reintroduced into the Iquitos, Loreto, Peru area during the early 1990s, where it displaced other anophelines and caused a major malaria epidemic. Since then, case numbers in Loreto have fluctuated, but annual increases have been reported since 2012.MethodsThe population genetic structure of An. darlingi sampled before and after the introduction of long-lasting insecticidal nets (LLINs) was investigated to test the hypothesis of temporal population change (2006 vs. 2012). Current samples of An. darlingi were used to test the hypothesis of ecological adaptation to human modified (highway) compared with wild (riverine) habitat, linked to forest cover. In total, 693 An. darlingi from nine localities in Loreto, Peru area were genotyped using 13 microsatellite loci. To test the hypothesis of habitat differentiation in An. darlingi biting time patterns, HBR and EIR, four collections of An. darlingi from five localities (two riverine and three highway) were analysed.ResultsAnalyses of microsatellite loci from seven (2006) and nine settlements (2012–2014) in the Iquitos area detected two distinctive populations with little overlap, although it is unclear whether this population replacement event is associated with LLIN distribution or climate. Within the 2012–2014 population two admixed subpopulations, A and B, were differentiated by habitat, with B significantly overrepresented in highway, and both in near-equal proportions in riverine. Both subpopulations had a signature of expansion and there was moderate genetic differentiation between them. Habitat and forest cover level had significant effects on HBR, such that Plasmodium transmission risk, as measured by EIR, in peridomestic riverine settlements was threefold higher than in peridomestic highway settlements. HBR was directly associated with available host biomass rather than forest cover.ConclusionsA population replacement event occurred between 2006 and 2012–2014, concurrently with LLIN distribution and a moderate El Niño event, and prior to an increase in malaria incidence. The likely drivers of this replacement cannot be determined with current data. The present-day An. darlingi population is composed of two highly admixed subpopulations, which appear to be in an early stage of differentiation, triggered by anthropogenic alterations to local habitat.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0863-4) contains supplementary material, which is available to authorized users.

Active Compounds Against Anopheles minimus Carboxypeptidase B for Malaria Transmission-Blocking Strategy.

Malaria transmission-blocking compounds have been studied to block the transmission of malaria parasites, especially the drug-resistant Plasmodium. Carboxypeptidase B (CPB) in the midgut of Anopheline mosquitoes has been demonstrated to be essential for the sexual development of Plasmodium in the mosquito. Thus, the CPB is a potential target for blocking compounds. The aim of this research was to screen compounds from the National Cancer Institute (NCI) diversity dataset and U.S. Food and Drug Administration (FDA)-approved drugs that could reduce the Anopheles CPB activity. The cDNA fragment of cpb gene from An. minimus (cpbAmi) was amplified and sequenced. The three-dimensional structure of CPB was predicted from the deduced amino acid sequence. The virtual screening of the compounds from NCI diversity set IV and FDA-approved drugs was performed against CPBAmi. The inhibition activity against CPBAmi of the top-scoring molecules was characterized invitro. Three compounds-NSC-1014, NSC-332670, and aminopterin with IC50 at 0.99 mM, 1.55 mM, and 0.062 mM, respectively-were found to significantly reduce the CPBAmi activity.

Plasmodium falciparum Gametocyte-Specific Antibody Profiling Reveals Boosting through Natural Infection and Identifies Potential Markers of Gametocyte Exposure.

Malaria elimination efforts would benefit from vaccines that block transmission of Plasmodium falciparum gametocytes from humans to mosquitoes. A clear understanding of gametocyte-specific antibody responses in exposed populations could help determine whether transmission-blocking vaccines (TBV) would be boosted by natural gametocyte exposure, and also inform the development of serologic tools to monitor gametocyte exposure in populations targeted for malaria elimination. To this end, plasma was collected from Malian children and adults before and after the 6-month malaria season and probed against a microarray containing 1,204 P. falciparum proteins. Using publicly available proteomic data, we classified 91 proteins as gametocyte specific and 69 as proteins not expressed by gametocytes. The overall breadth and magnitude of gametocyte-specific IgG responses increased during the malaria season, although they were consistently lower than IgG responses to nongametocyte antigens. Notably, IgG specific for the TBV candidates Pfs48/45 and Pfs230 increased during the malaria season. In addition, IgGs specific for the gametocyte proteins Pfmdv1, Pfs16, PF3D7_1346400, and PF3D7_1024800 were detected in nearly all subjects, suggesting that seroconversion to these proteins may be a sensitive indicator of gametocyte exposure, although further studies are needed to determine the specificity and kinetics of these potential serologic markers. These findings suggest that TBV-induced immunity would be boosted through natural gametocyte exposure, and that antibody responses to particular antigens may reliably indicate gametocyte exposure.

Plasmodium transmission blocking activities of Vernonia amygdalina extracts and isolated compounds.

BackgroundMedicinal plants are a validated source for discovery of new leads and standardized herbal medicines. The aim of this study was to assess the activity of Vernoniaamygdalina leaf extracts and isolated compounds against gametocytes and sporogonic stages of Plasmodiumberghei and to validate the findings on field isolates of Plasmodium falciparum.MethodsAqueous (Ver-H2O) and ethanolic (Ver-EtOH) leaf extracts were tested in vivo for activity against sexual and asexual blood stage P. berghei parasites. In vivo transmission blocking effects of Ver-EtOH and Ver-H2O were estimated by assessing P. berghei oocyst prevalence and density in Anopheles stephensi mosquitoes. Activity targeting early sporogonic stages (ESS), namely gametes, zygotes and ookinetes was assessed in vitro using P. berghei CTRPp.GFP strain. Bioassay guided fractionation was performed to characterize V.amygdalina fractions and molecules for anti-ESS activity. Fractions active against ESS of the murine parasite were tested for ex vivo transmission blocking activity on P.falciparum field isolates. Cytotoxic effects of extracts and isolated compounds vernolide and vernodalol were evaluated on the human cell lines HCT116 and EA.hy926.ResultsVer-H2O reduced the P. berghei macrogametocyte density in mice by about 50% and Ver-EtOH reduced P. berghei oocyst prevalence and density by 27 and 90%, respectively, in An.stephensi mosquitoes. Ver-EtOH inhibited almost completely (>90%) ESS development in vitro at 50 μg/mL. At this concentration, four fractions obtained from the ethylacetate phase of the methanol extract displayed inhibitory activity >90% against ESS. Three tested fractions were also found active against field isolates of the human parasite P. falciparum, reducing oocyst prevalence in Anopheles coluzzii mosquitoes to one-half and oocyst density to one-fourth of controls. The molecules and fractions displayed considerable cytotoxicity on the two tested cell-lines.ConclusionsVernonia amygdalina leaves contain molecules affecting multiple stages of Plasmodium, evidencing its potential for drug discovery. Chemical modification of the identified hit molecules, in particular vernodalol, could generate a library of druggable sesquiterpene lactones. The development of a multistage phytomedicine designed as preventive treatment to complement existing malaria control tools appears a challenging but feasible goal.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0812-2) contains supplementary material, which is available to authorized users.

Identifying avian malaria vectors: sampling methods influence outcomes.

BackgroundThe role of vectors in the transmission of avian malaria parasites is currently understudied. Many studies that investigate parasite-vector relationships use limited trapping techniques and/or identify potential competent vectors in the field in such ways that cannot distinguish between an infected or infectious vector. Without the use of multiple trapping techniques that address the specific biology of diverse mosquito species, and without looking at the infection status of individual mosquitoes, it is not possible to make dependable conclusions on the role of mosquitoes in the transmission of avian malaria parasites.MethodsWe conducted two years of mosquito collections at a riparian preserve in California where a wide diversity of species were collected with multiple trap types. We hypothesized that competent mosquito species can influence the distribution and diversity of avian malaria parasites by acting as a compatibility filter for specific Plasmodium species. To determine the infection status of all individual mosquitoes for Plasmodium species/lineages, amplification within the cytochrome b gene was carried out on over 3000 individual mosquito thoraxes, and for those that tested positive we then repeated the same process for abdomens and salivary glands.ResultsOur data show heterogeneity in the transmissibility of Plasmodium among ornithophillic mosquito species. More specifically, Culex stigmatosoma appears to not be a vector of Plasmodium homopolare, a parasite that is prevalent in the avian population, but is a vector of multiple other Plasmodium species/lineages.ConclusionsOur results suggest that conclusions made on the role of vectors from studies that do not use different mosquito trapping methods should be re-evaluated with caution, as we documented the potential for trapping biases, which may cause studies to miss important roles of specific mosquito species in the transmission of avian malaria. Moreover, we document heterogeneity in the transmission of Plasmodium spp. by mosquitoes can influence Plasmodium diversity and prevalence in specific locations to Plasmodium-vector incompatibilities.

Proteomic changes occurring in the malaria mosquitoes Anopheles gambiae and Anopheles stephensi during aging

The age of mosquitoes is a crucial determinant of their ability to transmit pathogens and their resistance to insecticides. We investigated changes to the abundance of proteins found in heads and thoraces of the malaria mosquitoes Anopheles gambiae and Anopheles stephensi as they aged. Protein expression changes were assessed using two-dimensional difference gel electrophoresis and the identity of differentially expressed proteins was determined by using either matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry or capillary high-pressure liquid chromatography coupled with a linear ion-trap (LTQ)-Orbitrap XL hybrid mass spectrometer. Protein biomarkers were validated by semi quantitative Western blot analysis.Nineteen and nine age dependent protein spots were identified for A. stephensi and A. gambiae, respectively. Among the proteins down-regulated with age were homologs of ADF/Cofilin, cytochome c1, heat shock protein-70 and eukaryotic translation initiation factor 5A (eIF5a). Proteins up-regulated with age included probable methylmalonate-semialdehyde dehydrogenase, voltage-dependent anion-selective channel and fructose bisphosphate aldolase. Semi quantitative Western blot analysis confirmed expression patterns observed by 2-D DIGE for eIF5a and ADF/Cofilin. Further work is recommended to determine whether these biomarkers are robust to infection, blood feeding and insecticide resistance. Robust biomarkers could then be incorporated into rapid diagnostic assays for ecological and epidemiological studies. Biological significanceIn this study, we have identified several proteins with characteristic changes in abundance in both A. gambiae and A. stephensi during their aging process. These changes may highlight underlying mechanisms beneath the relationship between mosquito age and factors affecting Plasmodium transmission and mosquito control. The similarity of changes in protein abundance between these species and the primary dengue vector Aedes aegypti, has revealed conserved patterns of aging-specific protein regulation.

No evidence for ape Plasmodium infections in humans in Gabon.

African great apes are naturally infected by a multitude of Plasmodium species most of them recently discovered, among which several are closely related to human malaria agents. However, it is still unknown whether these animals can serve as source of infections for humans living in their vicinity. To evaluate this possibility, we analysed the nature of Plasmodium infections from a bank of 4281 human blood samples collected in 210 villages of Gabon, Central Africa. Among them, 2255 were detected positive to Plasmodium using molecular methods (Plasmodium Cytochrome b amplification). A high throughput sequencing technology (454 GS-FLX Titanium technology, Roche) was then used to identify the Plasmodium species present within each positive sample. Overall, we identified with confidence only three species infecting humans in Gabon: P. falciparum, P. malariae and P. ovale. None of the species known to infect non-human primates in Central Africa was found. Our study shows that ape Plasmodium parasites of the subgenus Laverania do not constitute a frequent source of infection for humans. It also suggests that some strong host genetic barriers must exist to prevent the cross species transmission of ape Plasmodium in a context of ever increasing contacts between humans and wildlife.

Gammaherpesvirus Co-infection with Malaria Suppresses Anti-parasitic Humoral Immunity.

Immunity to non-cerebral severe malaria is estimated to occur within 1-2 infections in areas of endemic transmission for Plasmodium falciparum. Yet, nearly 20% of infected children die annually as a result of severe malaria. Multiple risk factors are postulated to exacerbate malarial disease, one being co-infections with other pathogens. Children living in Sub-Saharan Africa are seropositive for Epstein Barr Virus (EBV) by the age of 6 months. This timing overlaps with the waning of protective maternal antibodies and susceptibility to primary Plasmodium infection. However, the impact of acute EBV infection on the generation of anti-malarial immunity is unknown. Using well established mouse models of infection, we show here that acute, but not latent murine gammaherpesvirus 68 (MHV68) infection suppresses the anti-malarial humoral response to a secondary malaria infection. Importantly, this resulted in the transformation of a non-lethal P. yoelii XNL infection into a lethal one; an outcome that is correlated with a defect in the maintenance of germinal center B cells and T follicular helper (Tfh) cells in the spleen. Furthermore, we have identified the MHV68 M2 protein as an important virus encoded protein that can: (i) suppress anti-MHV68 humoral responses during acute MHV68 infection; and (ii) plays a critical role in the observed suppression of anti-malarial humoral responses in the setting of co-infection. Notably, co-infection with an M2-null mutant MHV68 eliminates lethality of P. yoelii XNL. Collectively, our data demonstrates that an acute gammaherpesvirus infection can negatively impact the development of an anti-malarial immune response. This suggests that acute infection with EBV should be investigated as a risk factor for non-cerebral severe malaria in young children living in areas endemic for Plasmodium transmission.

CAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission.

Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites.

Risk factors for Plasmodium falciparum gametocyte positivity in a longitudinal cohort.

Malaria transmission intensity is highly heterogeneous even at a very small scale. Implementing targeted intervention in malaria transmission hotspots offers the potential to reduce the burden of disease both locally and in adjacent areas. Transmission of malaria parasites from man to mosquito requires the production of gametocyte stage parasites. Cluster analysis of a 19-year long cohort study for gametocyte carriage revealed spatially defined gametocyte hotspots that occurred during the time when chloroquine was the drug used for clinical case treatment. In addition to known risk factors for gametocyte carriage, notably young age (<15 years old) and associated with a clinical episode, blood groups B and O increased risk compared to groups A and AB. A hotspot of clinical P. falciparum clinical episodes that overlapped the gametocyte hotspots was also identified. Gametocyte positivity was found to be increased in individuals who had been treated with chloroquine, as opposed to other drug treatment regimens, for a clinical P. falciparum episode up to 30 days previously. It seems likely the hotspots were generated by a vicious circle of ineffective treatment of clinical cases and concomitant gametocyte production in a sub-population characterized by an increased prevalence of all the identified risk factors. While rapid access to treatment with an effective anti-malarial can reduce the duration of gametocyte carriage and onward parasite transmission, localised hotspots represent a challenge to malaria control and eventual eradication.

Cyclic GMP balance is critical for malaria parasite transmission from the mosquito to the mammalian host.

ABSTRACTTransmission of malaria occurs during Anopheles mosquito vector blood meals, when Plasmodium sporozoites that have invaded the mosquito salivary glands are delivered to the mammalian host. Sporozoites display a unique form of motility that is essential for their movement across cellular host barriers and invasion of hepatocytes. While the molecular machinery powering motility and invasion is increasingly well defined, the signaling events that control these essential parasite activities have not been clearly delineated. Here, we identify a phosphodiesterase (PDEγ) in Plasmodium, a regulator of signaling through cyclic nucleotide second messengers. Reverse transcriptase PCR (RT-PCR) analysis and epitope tagging of endogenous PDEγ detected its expression in blood stages and sporozoites of Plasmodium yoelii. Deletion of PDEγ (pdeγ−) rendered sporozoites nonmotile, and they failed to invade the mosquito salivary glands. Consequently, PDEγ deletion completely blocked parasite transmission by mosquito bite. Strikingly, pdeγ− sporozoites showed dramatically elevated levels of cyclic GMP (cGMP), indicating that a perturbation in cyclic nucleotide balance is involved in the observed phenotypic defects. Transcriptome sequencing (RNA-Seq) analysis of pdeγ− sporozoites revealed reduced transcript abundance of genes that encode key components of the motility and invasion apparatus. Our data reveal a crucial role for PDEγ in maintaining the cyclic nucleotide balance in the malaria parasite sporozoite stage, which in turn is essential for parasite transmission from mosquito to mammal.

Recombinant Pvs48/45 antigen expressed in E. coli generates antibodies that block malaria transmission in Anopheles albimanus mosquitoes.

Transmission of malaria parasites from humans to Anopheles mosquitoes can be inhibited by specific antibodies elicited during malaria infection, which target surface Plasmodium gametocyte/gamete proteins. Some of these proteins may have potential for vaccine development. Pvs48/45 is a P. vivax gametocyte surface antigen orthologous to Pfs48/45, which may play a role during parasite fertilization and thus has potential for transmission blocking (TB) activity. Here we describe the expression of a recombinant Pvs48/45 protein expressed in Escherichia coli as a ∼60kDa construct which we tested for antigenicity using human sera and for its immunogenicity and transmission blocking activity of specific anti-mouse and anti-monkey Pvs48/45 antibodies. The protein reacted with sera of individuals from malaria-endemic areas and in addition induced specific IgG antibody responses in BALB/c mice and Aotus l. griseimembra monkeys. Sera from both immunized animal species recognized native P. vivax protein in Western blot (WB) and immunofluorescence assays. Moreover, sera from immunized mice and monkeys produced significant inhibition of parasite transmission to An. Albimanus mosquitoes as shown by membrane feeding assays. Results indicate the presence of reactive epitopes in the Pvs48/45 recombinant product that induce antibodies with TB activity. Further testing of this protein is ongoing to determine its vaccine potential.

Alga-produced malaria transmission-blocking vaccine candidate Pfs25 formulated with a human use-compatible potent adjuvant induces high-affinity antibodies that block Plasmodium falciparum infection of mosquitoes.

A vaccine to prevent the transmission of malaria parasites from infected humans to mosquitoes is an important component for the elimination of malaria in the 21st century, yet it remains neglected as a priority of malaria vaccine development. The lead candidate for Plasmodium falciparum transmission-blocking vaccine development, Pfs25, is a sexual stage surface protein that has been produced for vaccine testing in a variety of heterologous expression systems. Any realistic malaria vaccine will need to optimize proper folding balanced against cost of production, yield, and potentially reactogenic contaminants. Here Chlamydomonas reinhardtii microalga-produced recombinant Pfs25 protein was formulated with four different human-compatible adjuvants (alum, Toll-like receptor 4 [TLR-4] agonist glucopyranosal lipid A [GLA] plus alum, squalene-oil-in-water emulsion, and GLA plus squalene-oil-in-water emulsion) and compared for their ability to induce malaria transmission-blocking antibodies. Alga-produced recombinant Pfs25 plus GLA plus squalene-oil-in-water adjuvant induced the highest titer and avidity in IgG antibodies, measured using alga-produced recombinant Pfs25 as the enzyme-linked immunosorbent assay (ELISA) antigen. These antibodies specifically reacted with the surface of P. falciparum macrogametes and zygotes and effectively prevented parasites from developing within the mosquito vector in standard membrane feeding assays. Alga-produced Pfs25 in combination with a human-compatible adjuvant composed of a TLR-4 agonist in a squalene-oil-in-water emulsion is an attractive new vaccine candidate that merits head-to-head comparison with other modalities of vaccine production and administration.

A spatial individual-based model predicting a great impact of copious sugar sources and resting sites on survival of Anopheles gambiae and malaria parasite transmission.

BackgroundAgent-based modelling (ABM) has been used to simulate mosquito life cycles and to evaluate vector control applications. However, most models lack sugar-feeding and resting behaviours or are based on mathematical equations lacking individual level randomness and spatial components of mosquito life. Here, a spatial individual-based model (IBM) incorporating sugar-feeding and resting behaviours of the malaria vector Anopheles gambiae was developed to estimate the impact of environmental sugar sources and resting sites on survival and biting behaviour.MethodsA spatial IBM containing An. gambiae mosquitoes and humans, as well as the village environment of houses, sugar sources, resting sites and larval habitat sites was developed. Anopheles gambiae behaviour rules were attributed at each step of the IBM: resting, host seeking, sugar feeding and breeding. Each step represented one second of time, and each simulation was set to run for 60 days and repeated 50 times. Scenarios of different densities and spatial distributions of sugar sources and outdoor resting sites were simulated and compared.ResultsWhen the number of natural sugar sources was increased from 0 to 100 while the number of resting sites was held constant, mean daily survival rate increased from 2.5% to 85.1% for males and from 2.5% to 94.5% for females, mean human biting rate increased from 0 to 0.94 bites per human per day, and mean daily abundance increased from 1 to 477 for males and from 1 to 1,428 for females. When the number of outdoor resting sites was increased from 0 to 50 while the number of sugar sources was held constant, mean daily survival rate increased from 77.3% to 84.3% for males and from 86.7% to 93.9% for females, mean human biting rate increased from 0 to 0.52 bites per human per day, and mean daily abundance increased from 62 to 349 for males and from 257 to 1120 for females. All increases were significant (P < 0.01). Survival was greater when sugar sources were randomly distributed in the whole village compared to clustering around outdoor resting sites or houses.ConclusionsIncreases in densities of sugar sources or outdoor resting sites significantly increase the survival and human biting rates of An. gambiae mosquitoes. Survival of An. gambiae is more supported by random distribution of sugar sources than clustering of sugar sources around resting sites or houses. Density and spatial distribution of natural sugar sources and outdoor resting sites modulate vector populations and human biting rates, and thus malaria parasite transmission.