Search

After being ingested by a female Anopheles mosquito during a bloodmeal on an infected host, and before they can reach the mosquito salivary glands to be transmitted to a new host, Plasmodium parasites must establish an infection of the mosquito midgut in the form of oocysts. To achieve this, they must first survive a series of robust innate immune responses that take place prior to, during, and immediately after ookinete traversal of the midgut epithelium. Understanding how parasites may evade these responses could highlight new ways to block malaria transmission. We show that an ookinete and sporozoite surface protein designated as PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43) is required for parasite evasion of the Anopheles coluzzii complement-like response. Disruption of PIMMS43 in the rodent malaria parasite Plasmodium berghei triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing components of the complement-like system through RNAi largely restores ookinete-to-oocyst transition but oocysts remain small in size and produce a very small number of sporozoites that additionally are not infectious, indicating that PIMMS43 is also essential for sporogonic development in the oocyst. Antibodies that bind PIMMS43 interfere with parasite immune evasion when ingested with the infectious blood meal and significantly reduce the prevalence and intensity of infection. PIMMS43 genetic structure across African Plasmodium falciparum populations indicates allelic adaptation to sympatric vector populations. These data add to our understanding of mosquito-parasite interactions and identify PIMMS43 as a target of malaria transmission blocking. [ABSTRACT FROM AUTHOR]

Background: Transmission-blocking interventions (TBIs) aim to eliminate malaria by reducing transmission of the parasite between the host and the invertebrate vector. TBIs include transmission-blocking drugs and vaccines that, when given to humans, are taken up by mosquitoes and inhibit parasitic development within the vector. Accurate methodologies are key to assess TBI efficacy to ensure that only the most potent candidates progress to expensive and time-consuming clinical trials. Measuring intervention efficacy can be problematic because there is substantial variation in the number of parasites in both the host and vector populations, which can impact transmission even in laboratory settings. Methods: A statistically robust empirical method is introduced for estimating intervention efficacy from standardised population assay experiments. This method will be more reliable than simple summary statistics as it captures changes in parasite density in different life-stages. It also allows efficacy estimates at a finer resolution than previous methods enabling the impact of the intervention over successive generations to be tracked. A major advantage of the new methodology is that it makes no assumptions on the population dynamics of infection. This enables both host-to-vector and vector-to-host transmission to be density-dependent (or other) processes and generates easy-tounderstand estimates of intervention efficacy. Results: This method increases the precision of intervention efficacy estimates and demonstrates that relying on changes in infection prevalence (the proportion of infected hosts) alone may be insufficient to capture the impact of TBIs, which also suppress parasite density in secondarily infected hosts. Conclusions: The method indicates that potentially useful, partially effective TBIs may require multiple infection cycles before substantial reductions in prevalence are observed, despite more rapidly suppressing parasite density. Accurate models to quantify efficacy will have important implications for understanding how TBI candidates might perform in field situations and how they should be evaluated in clinical trials. [ABSTRACT FROM AUTHOR]

Over a century since Ronald Ross discovered that malaria is caused by the bite of an infectious mosquito it is still unclear how the number of parasites injected influences disease transmission. Currently it is assumed that all mosquitoes with salivary gland sporozoites are equally infectious irrespective of the number of parasites they harbour, though this has never been rigorously tested. Here we analyse >1000 experimental infections of humans and mice and demonstrate a dose-dependency for probability of infection and the length of the host pre-patent period. Mosquitoes with a higher numbers of sporozoites in their salivary glands following blood-feeding are more likely to have caused infection (and have done so quicker) than mosquitoes with fewer parasites. A similar dose response for the probability of infection was seen for humans given a pre-erythrocytic vaccine candidate targeting circumsporozoite protein (CSP), and in mice with and without transfusion of anti-CSP antibodies. These interventions prevented infection more efficiently from bites made by mosquitoes with fewer parasites. The importance of parasite number has widespread implications across malariology, ranging from our basic understanding of the parasite, how vaccines are evaluated and the way in which transmission should be measured in the field. It also provides direct evidence for why the only registered malaria vaccine RTS,S was partially effective in recent clinical trials. [ABSTRACT FROM AUTHOR]

Gametocytes are the sole P lasmodium parasite stages that infect mosquitoes; therefore development of functional gametes is required for malaria transmission. Flagellum assembly of the P lasmodium male gamete differs from that of most other eukaryotes in that it is intracytoplasmic but retains a key conserved feature: axonemes assemble from basal bodies. The centriole/basal body protein SAS-6 normally regulates assembly and duplication of these organelles and its depletion causes severe flagellar/ciliary abnormalities in a diverse array of eukaryotes. Since basal body and flagellum assembly are intimately coupled to male gamete development in P lasmodium, we hypothesized that SAS-6 disruption may cause gametogenesis defects and perturb transmission. We show that P lasmodium berghei sas6 knockouts display severely abnormal male gametogenesis presenting reduced basal body numbers, axonemal assembly defects and abnormal nuclear allocation. The defects in gametogenesis reduce fertilization and render Pbsas6 knockouts less infectious to mosquitoes. Additionally, we show that lack of Pbsas6 blocks transmission from mosquito to vertebrate host, revealing an additional yet undefined role in ookinete to sporulating oocysts transition. These findings underscore the vulnerability of the basal body/ SAS-6 to malaria transmission blocking interventions. [ABSTRACT FROM AUTHOR]

Since 2010 two global reviews of malaria research have recognized that local elimination and eradication of Plasmodium parasites are key drivers for further experimentation. To achieve these ambitious objectives it is universally recognized we must reduce malaria transmission through the mosquito vectors. A plethora of new laboratory assays are being developed to interrogate malaria transmission from the gametocyte to the sporozoite stage: assays that augment well-established field protocols to determine the entomological inoculation rate. However, the diverse readouts of these assays are not directly comparable. Here we attempt to identify the utility of each assay and provide rational frameworks by which to compare the impacts recorded by the diverse methodologies. [ABSTRACT FROM AUTHOR]

Abstract: The standard membrane feeding assay (SMFA) is currently considered to be the ‘gold standard’ for assessing the effectiveness of malaria transmission blocking interventions (TBIs) in vivo. The operation and analysis of SMFAs has varied between laboratories: field scientists often measure TBI efficacy as a reduction in the prevalence of infected mosquitoes whilst laboratory scientists are more likely to quote efficacy as a change in the number of oocysts within the mosquito. These metrics give outputs that differ widely, resulting in a need for greater understanding of how the SMFA informs TBI assessment. Using data from 536 different assays (conducted on Plasmodium falciparum and Plasmodium berghei, in either Anopheles gambiae or Anopheles stephensi) it is shown that the relationship between these metrics is complex, yet predictable. Results demonstrate that the distribution of oocysts between mosquitoes is highly aggregated, making efficacy estimates based on reductions in intensity highly uncertain. Analysis of 30 SMFAs carried out on the same TBI confirms that the observed reduction in prevalence depends upon the parasite exposure (as measured by oocyst intensity in the control group), with assays which have lower exposure appearing more effective. By contrast, if efficacy is estimated as a reduction in oocyst intensity, then this candidate demonstrated constant efficacy, irrespective of the exposure level. To report transmission-blockade efficacy accurately, the results of SMFAs should give both the prevalence and intensity of oocysts in both the control and intervention group. Candidates should be assessed against a range of parasite exposures to allow laboratory results to be extrapolated to different field situations. Currently, many studies assessing TBIs are underpowered and uncertainties in efficacy estimates rarely reported. Statistical techniques that account for oocyst over-dispersion can reduce the number of mosquitoes that need to be dissected and allow TBI candidates from different laboratories to be accurately compared. [Copyright &y& Elsevier]

Summary The malaria life cycle relies on the successful transfer of the parasite between its human and mosquito hosts. We identified a Plasmodium berghei secreted protein (PBANKA_131270) that plays distinct roles in both the mammal-to-mosquito and the mosquito-to-mammal transitions. This protein, here named gamete egress and sporozoite traversal (GEST), plays an important role in the egress of male and female gametes from the vertebrate red blood cell. Interestingly, GEST is also required following the bite of the infected mosquito, for sporozoite progression through the skin. We found PbGEST to be secreted shortly after activation of the intraerythrocytic gametocyte, and during sporozoite migration. These findings indicate that a single malaria protein may have pleiotropic roles in different parasites stages mediating transmission between its insect and mammalian hosts. [ABSTRACT FROM AUTHOR]

Abstract: Fertilization in Plasmodium is a complex process that occurs in the gut of the female Anopheles mosquito upon uptake of a bloodmeal. It requires the emergence of the gametocyte from the RBC and release of eight flagellate male gametes from each male cell, and subsequent fertilization of a similarly emerged immotile extracellular female macrogamete. Previous studies have demonstrated that antibodies against male gamete surface proteins ingested from the blood of an infected and immunized host inhibit parasite transmission. Gene disruption studies in Plasmodium berghei and complimentary studies on the green alga Chlamydomonas have shown that a conserved male gamete sterility gene, HAP2, is essential for fusion of male and female gametes. Genetic disruption of the HAP2 locus revealed that parasite fertilization is prevented, yet hap2 KO male gametes still retained the ability to form tight pre-fusion membrane attachments with females.We demonstrate that heterologous expression of the P. berghei HAP2 protein in Escherichia coli, and subsequent immunization of rabbits, has produced anti-sera that react specifically with recombinant HAP2, and with the native protein on the male gamete. Additionally, anti-HAP2 sera reduces in vitro formation of ookinetes by up to 81%, and, using standard membrane feeding assays, reduces oocyst burden within the mosquito host by up to 81.1%, and prevalence of in vivo infection by up to 34%. Inhibition is dose dependent. These results indicate that HAP2 should be considered as a potential target for any future anti-malarial transmission-blocking vaccine. [Copyright &y& Elsevier]

The evaluation of transmission reducing interventions (TRI) to control malaria widely uses membrane feeding assays. In such assays, the intensity of Plasmodium infection in the vector might affect the measured efficacy of the candidates to block transmission. Gametocyte density in the host blood is a determinant of the infection success in the mosquito, however, uncertain estimates of parasite densities and intrinsic characteristics of the infected blood can induce variability. To reduce this variation, a feasible method is to dilute infectious blood samples. We describe the effect of diluting samples of Plasmodium -containing blood samples to allow accurate relative measures of gametocyte densities and their impact on mosquito infectivity and TRI efficacy. Natural Plasmodium falciparum samples were diluted to generate a wide range of parasite densities, and fed to Anopheles coluzzii mosquitoes. This was compared with parallel dilutions conducted on Plasmodium berghei infections. We examined how blood dilution influences the observed blocking activity of anti-Pbs28 monoclonal antibody using the P. berghei/Anopheles stephensi system. In the natural species combination P. falciparum/An. coluzzii , blood dilution using heat-inactivated, infected blood as diluents, revealed positive near linear relationships, between gametocyte densities and oocyst loads in the range tested. A similar relationship was observed in the P. berghei/An. stephensi system when using a similar dilution method. In contrast, diluting infected mice blood with fresh uninfected blood dramatically increases the infectiousness. This suggests that highly infected mice blood contains inhibitory factors or reduced blood moieties, which impede infection and may in turn, lead to misinterpretation when comparing individual TRI evaluation assays. In the lab system, the transmission blocking activity of an antibody specific for Pbs28 was confirmed to be density-dependent. This highlights the need to carefully interpret evaluations of TRI candidates, regarding gametocyte densities in the P. berghei/An. stephensi system. [ABSTRACT FROM AUTHOR]

Abstract: The transformation of malaria ookinetes into oocysts occurs in the mosquito midgut and is a major bottleneck for parasite transmission. The secreted ookinete surface protein, circumsporozoite- and thrombospondin-related adhesive protein (TRAP)-related protein (CTRP), is essential for this transition and hence constitutes a potential target for malaria transmission blockade. CTRP is a modular multidomain protein containing six tandem von Willebrand factor A-like (A) domains and seven tandem thrombospondin type I repeat-like (TS) domains. Here we present, to our knowledge, the first structure–function analysis of CTRP using genetically modified Plasmodium berghei parasites expressing mutant versions of the ctrp gene. Our data show that the A domains of CTRP are critical for ookinete gliding motility and oocyst formation whilst, unexpectedly, its TS domains are fully redundant. These results may have important implications for the design of CTRP-based transmission blocking strategies. [Copyright &y& Elsevier]