Targeting the Parasite to Suppress Malaria Transmission.

This article attempts to draw together current knowledge on the biology of Plasmodium and experience gained from past control campaigns to interpret and guide current efforts to discover and develop exciting new strategies targeting the parasite with the objective of interrupting transmission. Particular note is made of the advantages of targeting often unappreciated small, yet vital, bottleneck populations to enhance both the impact and the useful lifetime of hard-won interventions. A case is made for the standardization of methods to measure transmission blockade to permit the rational comparison of how diverse interventions (drugs, vaccines, insecticides, Genetically Modified technologies) targeting disparate aspects of parasite biology may impact upon the commonly used parameter of parasite prevalence in the human population.

Transmission blocking potency and immunogenicity of a plant-produced Pvs25-based subunit vaccine against Plasmodium vivax.

Malaria transmission blocking (TB) vaccines (TBVs) directed against proteins expressed on the sexual stages of Plasmodium parasites are a potentially effective means to reduce transmission. Antibodies induced by TBVs block parasite development in the mosquito, and thus inhibit transmission to further human hosts. The ookinete surface protein P25 is a primary target for TBV development. Recently, transient expression in plants using hybrid viral vectors has demonstrated potential as a strategy for cost-effective and scalable production of recombinant vaccines. Using a plant virus-based expression system, we produced recombinant P25 protein of Plasmodium vivax (Pvs25) in Nicotiana benthamiana fused to a modified lichenase carrier protein. This candidate vaccine, Pvs25-FhCMB, was purified, characterized and evaluated for immunogenicity and efficacy using multiple adjuvants in a transgenic rodent model. An in vivo TB effect of up to a 65% reduction in intensity and 54% reduction in prevalence was observed using Abisco-100 adjuvant. The ability of this immunogen to induce a TB response was additionally combined with heterologous prime-boost vaccination with viral vectors expressing Pvs25. Significant blockade was observed when combining both platforms, achieving a 74% and 68% reduction in intensity and prevalence, respectively. This observation was confirmed by direct membrane feeding on field P. vivax samples, resulting in reductions in intensity/prevalence of 85.3% and 25.5%. These data demonstrate the potential of this vaccine candidate and support the feasibility of expressing Plasmodium antigens in a plant-based system for the production of TBVs, while demonstrating the potential advantages of combining multiple vaccine delivery systems to maximize efficacy.

Comparative assessment of transmission-blocking vaccine candidates against Plasmodium falciparum.

Malaria transmission-blocking vaccines (TBVs) target the development of Plasmodium parasites within the mosquito, with the aim of preventing malaria transmission from one infected individual to another. Different vaccine platforms, mainly protein-in-adjuvant formulations delivering the leading candidate antigens, have been developed independently and have reported varied transmission-blocking activities (TBA). Here, recombinant chimpanzee adenovirus 63, ChAd63, and modified vaccinia virus Ankara, MVA, expressing AgAPN1, Pfs230-C, Pfs25, and Pfs48/45 were generated. Antibody responses primed individually against all antigens by ChAd63 immunization in BALB/c mice were boosted by the administration of MVA expressing the same antigen. These antibodies exhibited a hierarchy of inhibitory activity against the NF54 laboratory strain of P. falciparum in Anopheles stephensi mosquitoes using the standard membrane feeding assay (SMFA), with anti-Pfs230-C and anti-Pfs25 antibodies giving complete blockade. The observed rank order of inhibition was replicated against P. falciparum African field isolates in A. gambiae in direct membrane feeding assays (DMFA). TBA achieved was IgG concentration dependent. This study provides the first head-to-head comparative analysis of leading antigens using two different parasite sources in two different vector species, and can be used to guide selection of TBVs for future clinical development using the viral-vectored delivery platform.

The cell biology of malaria infection of mosquito: advances and opportunities.

Recent reviews (Feachem et al.; Alonso et al.) have concluded that in order to have a sustainable impact on the global burden of malaria, it is essential that we knowingly reduce the global incidence of infected persons. To achieve this we must reduce the basic reproductive rate of the parasites to < 1 in diverse epidemiological settings. This can be achieved by impacting combinations of the following parameters: the number of mosquitoes relative to the number of persons, the mosquito/human biting rate, the proportion of mosquitoes carrying infectious sporozoites, the daily survival rate of the infectious mosquito and the ability of malaria-infected persons to infect mosquito vectors. This paper focuses on our understanding of parasite biology underpinning the last of these terms: infection of the mosquito. The article attempts to highlight central issues that require further study to assist in the discovery of useful transmission-blocking measures.

The Plasmodium berghei sexual stage antigen PSOP12 induces anti-malarial transmission blocking immunity both in vivo and in vitro.

Anti-malarial transmission-blocking vaccines (TBVs) aim to inhibit the transmission of Plasmodium from humans to mosquitoes by targeting the sexual/ookinete stages of the parasite. Successful use of such interventions will subsequently result in reduced cases of malarial infection within a human population, leading to local elimination. There are currently only five lead TBV candidates under examination. There is a consequent need to identify novel antigens to allow the formulation of new potent TBVs. Here we describe the design and evaluation of a potential TBV (BDES-PbPSOP12) targeting Plasmodium berghei PSOP12 based on the baculovirus dual expression system (BDES), enabling expression of antigens on the surface of viral particles and within infected mammalian cells. In silico studies have previously suggested that PSOP12 (Putative Secreted Ookinete Protein 12) is expressed within the sexual stages of the parasite (gametocytes, gametes and ookinetes), and is a member of the previously characterized 6-Cys family of plasmodial proteins. We demonstrate that PSOP12 is expressed within the sexual/ookinete forms of the parasite, and that sera obtained from mice immunized with BDES-PbPSOP12 can recognize the surface of the male and female gametes, and the ookinete stages of the parasite. Immunization of mice with BDES-PbPSOP12 confers modest but significant transmission-blocking activity in vivo by active immunization (53.1% reduction in oocyst intensity, 10.9% reduction in oocyst prevalence). Further assessment of transmission-blocking potency ex vivo shows a dose-dependent response, with up to a 76.4% reduction in intensity and a 47.2% reduction in prevalence observed. Our data indicates that PSOP12 in Plasmodium spp. could be a potential new TBV target candidate, and that further experimentation to examine the protein within human malaria parasites would be logical.

Lead clinical and preclinical antimalarial drugs can significantly reduce sporozoite transmission to vertebrate populations.

To achieve malarial elimination, we must employ interventions that reduce the exposure of human populations to infectious mosquitoes. To this end, numerous antimalarial drugs are under assessment in a variety of transmission-blocking assays which fail to measure the single crucial criteria of a successful intervention, namely impact on case incidence within a vertebrate population (reduction in reproductive number/effect size). Consequently, any reduction in new infections due to drug treatment (and how this may be influenced by differing transmission settings) is not currently examined, limiting the translation of any findings. We describe the use of a laboratory population model to assess how individual antimalarial drugs can impact the number of secondary Plasmodium berghei infections over a cycle of transmission. We examine the impact of multiple clinical and preclinical drugs on both insect and vertebrate populations at multiple transmission settings. Both primaquine (>6 mg/kg of body weight) and NITD609 (8.1 mg/kg) have significant impacts across multiple transmission settings, but artemether and lumefantrine (57 and 11.8 mg/kg), OZ439 (6.5 mg/kg), and primaquine (<1.25 mg/kg) demonstrated potent efficacy only at lower-transmission settings. While directly demonstrating the impact of antimalarial drug treatment on vertebrate populations, we additionally calculate effect size for each treatment, allowing for head-to-head comparison of the potential impact of individual drugs within epidemiologically relevant settings, supporting their usage within elimination campaigns.

A male and female gametocyte functional viability assay to identify biologically relevant malaria transmission-blocking drugs.

Malaria elimination will require interventions that prevent parasite transmission from the human host to the mosquito. Experimentally, this is usually determined by the expensive and laborious Plasmodium falciparum standard membrane feeding assay (PfSMFA), which has limited utility for high-throughput drug screening. In response, we developed the P. falciparum dual gamete formation assay (PfDGFA), which faithfully simulates the initial stages of the PfSMFA in vitro. It utilizes a dual readout that individually and simultaneously reports on the functional viability of male and female mature stage V gametocytes. To validate, we screen the Medicines for Malaria Venture (MMV) Malaria Box library with the PfDGFA. Unique to this assay, we find compounds that target male gametocytes only and also compounds with reversible and irreversible activity. Most importantly, we show that compound activity in the PfDGFA accurately predicts activity in PfSMFAs, which validates and supports its adoption into the transmission-stage screening pipeline.

Transmission-blocking interventions eliminate malaria from laboratory populations.

Transmission-blocking interventions aim to reduce the prevalence of infection in endemic communities by targeting Plasmodium within the insect host. Although many studies have reported the successful reduction of infection in the mosquito vector, direct evidence that there is an onward reduction in infection in the vertebrate host is lacking. Here we report the first experiments using a population, transmission-based study of Plasmodium berghei in Anopheles stephensi to assess the impact of a transmission-blocking drug upon both insect and host populations over multiple transmission cycles. We demonstrate that the selected transmission-blocking intervention, which inhibits transmission from vertebrate to insect by only 32%, reduces the basic reproduction number of the parasite by 20%, and in our model system can eliminate Plasmodium from mosquito and mouse populations at low transmission intensities. These findings clearly demonstrate that use of transmission-blocking interventions alone can eliminate Plasmodium from a vertebrate population, and have significant implications for the future design and implementation of transmission-blocking interventions within the field.

Proteomic analysis of Plasmodium in the mosquito: progress and pitfalls.

Here we discuss proteomic analyses of whole cell preparations of the mosquito stages of malaria parasite development (i.e. gametocytes, microgamete, ookinete, oocyst and sporozoite) of Plasmodium berghei. We also include critiques of the proteomes of two cell fractions from the purified ookinete, namely the micronemes and cell surface. Whereas we summarise key biological interpretations of the data, we also try to identify key methodological constraints we have met, only some of which we were able to resolve. Recognising the need to translate the potential of current genome sequencing into functional understanding, we report our efforts to develop more powerful combinations of methods for the in silico prediction of protein function and location. We have applied this analysis to the proteome of the male gamete, a cell whose very simple structural organisation facilitated interpretation of data. Some of the in silico predictions made have now been supported by ongoing protein tagging and genetic knockout studies. We hope this discussion may assist future studies.

The flagellum in malarial parasites.

The malarial parasites assemble flagella exclusively during the formation of the male gamete in the midgut of the female mosquito vector. The observation of gamete formation ex vivo reported by Laveran (Laveran MA: De la nature parasitaire des accidents de l'impaludisme. Comptes Rendues De La Societe de Biologie. Paris 1881, 93:627-630) was seminal to the discovery of the parasite itself. Following ingestion of malaria-infected blood by the mosquito, microgamete formation from the terminally arrested gametocytes is exceptionally rapid, completing three mitotic divisions in just a few minutes, and is precisely regulated. This review attempts to draw together the diverse original observations with subsequent electron microscopic studies, and recent work on the signalling pathways regulating sexual development, together with transcriptomic and proteomic studies that are paving the way to new understandings of the molecular mechanisms involved and the potential they offer for effective interventions to block the transmission of the parasites in natural communities.

Malaria, sexual development and transmission: retrospect and prospect.

It is difficult to recapture the excitement of recent research into the malaria parasites. Plasmodium has shown itself to be a most elegant, resourceful and downright devious cell. To reveal any of its manifold secrets is a hard-won privilege. The thrill of this intellectual endeavour, however, has to be tempered by the realism that we have made unremarkable progress in attacking malaria in the field, where it remains almost as omnipresent as it ever was in the 19th and 20th centuries, and both the parasite and vector have become more difficult to control than ever before. This personal view looks back at the significant progress made, and forward to the challenges of the future, focusing on work on sexual development.

A DNA prime-modified vaccinia virus ankara boost vaccine encoding thrombospondin-related adhesion protein but not circumsporozoite protein partially protects healthy malaria-naive adults against Plasmodium falciparum sporozoite challenge.

The safety, immunogenicity, and efficacy of DNA and modified vaccinia virus Ankara (MVA) prime-boost regimes were assessed by using either thrombospondin-related adhesion protein (TRAP) with a multiple-epitope string ME (ME-TRAP) or the circumsporozoite protein (CS) of Plasmodium falciparum. Sixteen healthy subjects who never had malaria (malaria-naive subjects) received two priming vaccinations with DNA, followed by one boosting immunization with MVA, with either ME-TRAP or CS as the antigen. Immunogenicity was assessed by ex vivo gamma interferon (IFN-gamma) enzyme-linked immunospot assay (ELISPOT) and antibody assay. Two weeks after the final vaccination, the subjects underwent P. falciparum sporozoite challenge, with six unvaccinated controls. The vaccines were well tolerated and immunogenic, with the DDM-ME TRAP regimen producing stronger ex vivo IFN-gamma ELISPOT responses than DDM-CS. One of eight subjects receiving the DDM-ME TRAP regimen was completely protected against malaria challenge, with this group as a whole showing significant delay to parasitemia compared to controls (P = 0.045). The peak ex vivo IFN-gamma ELISPOT response in this group correlated strongly with the number of days to parasitemia (P = 0.033). No protection was observed in the DDM-CS group. Prime-boost vaccination with DNA and MVA encoding ME-TRAP but not CS resulted in partial protection against P. falciparum sporozoite challenge in the present study.

A proteomic analysis of malaria biology: integration of old literature and new technologies.

The genomic revolution has brought a new vitality into research on Plasmodium, its insect and vertebrate hosts. At the cellular level nowhere is the impact greater than in the analysis of protein expression and the 'assembly' of the supramolecular machines that together comprise the functional cell. The repetitive phases of invasion and replication that typify the malaria life cycle, together with the unique phase of sexual differentiation provide a powerful platform on which to investigate the 'molecular machines' that underpin parasite strategy and stage-specific functions. This approach is illustrated here in an analysis of the ookinete of Plasmodium berghei. Such analyses are useful only if conducted with a secure understanding of parasite biology. The importance of carefully searching the older literature to reach this understanding cannot be over-emphasised. When viewed together, the old and new data can give rapid and penetrating insights into what some might now term the 'Systems-Biology' of Plasmodium.

Mosquito--malaria interactions: a reappraisal of the concepts of susceptibility and refractoriness.

This paper considers the available literature on the transmission of malaria by insects and concludes that, in contrast to the commonly held view (that implies mosquitoes are naturally vectors of malaria), it is more useful to consider that mosquitoes, like plants, normally express a variety of gene products, which together render the host resistant to infection. The consequences of this hypothesis upon current research are that when studying the passage of the malarial parasite through a competent vector it is relevant to ask either 'How have the natural innate defences of the insect failed?' or 'What mechanisms has the parasite used to overcome these defences?' At the population level, the hypothesis is consistent with the conclusions of Koella et al. that the evolutionary cost of maintaining defence mechanisms that can render the mosquito refractory (e.g. melanization) has prevented fixation of the necessary gene(s) in the insect population. We simply extend that concept by stating the innate and genetic defences that confer the natural (and sometimes incomplete) resistance to infection are of sustainable cost, with the consequence that the encoding genes may become highly prevalent or fixed in a population.

The dynamics of interactions between Plasmodium and the mosquito: a study of the infectivity of Plasmodium berghei and Plasmodium gallinaceum, and their transmission by Anopheles stephensi, Anopheles gambiae and Aedes aegypti.

Knowledge of parasite-mosquito interactions is essential to develop strategies that will reduce malaria transmission through the mosquito vector. In this study we investigated the development of two model malaria parasites, Plasmodium berghei and Plasmodium gallinaceum, in three mosquito species Anopheles stephensi, Anopheles gambiae and Aedes aegypti. New methods to study gamete production in vivo in combination with GFP-expressing ookinetes were employed to measure the large losses incurred by the parasites during infection of mosquitoes. All three mosquito species transmitted P. gallinaceum; P. berghei was only transmitted by Anopheles spp. Plasmodium gallinaceum initiates gamete production with high efficiency equally in the three mosquito species. By contrast P. berghei is less efficiently activated to produce gametes, and in Ae. aegypti microgamete formation is almost totally suppressed. In all parasite/vector combinations ookinete development is inefficient, 500-100,000-fold losses were encountered. Losses during ookinete-to-oocyst transformation range from fivefold in compatible vector parasite combinations (P. berghei/An. stephensi), through >100-fold in poor vector/parasite combinations (P. gallinaceum/An. stephensi), to complete blockade (>1,500 fold) in others (P. berghei/Ae. aegypti). Plasmodium berghei ookinetes survive poorly in the bloodmeal of Ae. aegypti and are unable to invade the midgut epithelium. Cultured mature ookinetes of P. berghei injected directly into the mosquito haemocoele produced salivary gland sporozoites in An. stephensi, but not in Ae. aegypti, suggesting that further species-specific incompatibilities occur downstream of the midgut epithelium in Ae. aegypti. These results show that in these parasite-mosquito combinations the susceptibility to malarial infection is regulated at multiple steps during the development of the parasites. Understanding these at the molecular level may contribute to the development of rational strategies to reduce the vector competence of malarial vectors.

Molecular interactions between Plasmodium and its insect vectors.

Our understanding of the intricate interactions between the malarial parasite and the mosquito vector is complicated both by the number and diversity of parasite and vector species, and by the experimental inaccessibility of phenomena under investigation. Steady developments in techniques to study the parasite in the mosquito have recently been augmented by methods to culture in their entirety the sporogonic stages of some parasite species. These, together with the new saturation technologies, and genetic transformation of both parasite and vector will permit penetrating studies into an exciting and largely unknown area of parasite-host interactions, an understanding of which must result in the development of new intervention strategies. This microreview highlights key areas of current basic molecular interest, and identifies numerous lacunae in our knowledge that must be filled if we are to make rational decisions for future control strategies. It will conclude by trying to explain why in the opinion of this reviewer understanding malaria-mosquito interactions may be critical to our future attempts to limit a disease of growing global importance.

Cryofracture electron microscopy of the ookinete pellicle of Plasmodium gallinaceum reveals the existence of novel pores in the alveolar membranes.

The malaria parasite invades the midgut tissue of its mosquito host as a motile form called the ookinete. We have examined the pellicle of the ookinete of Plasmodium gallinaceum by freeze-fracture and quick-freeze, deep-etch electron microscopy. The general organization is analogous to that of invasive stages of other members of Apicomplexa. The pellicle is composed of three membranes: the plasma membrane, and the two linked intermediate and inner membranes, which in the ookinete form one flattened vacuole that is located beneath the plasma membrane. The edges of this vacuole form a longitudinal suture. Beneath the vacuole is found an array of microtubules that are connected to the inner membrane by intramembranous particles. During freeze-fracture, the membranes can split along their hydrophobic planes, thus yielding six fracture faces, each of which displays a characteristic pattern of intramembranous particles. Additionally, we find that the ookinete pellicle differs from all other apicomplexan motile stages by the presence of large pores. These pores are of unknown function, but clearly might constitute a novel pathway for the transport of molecules to and from the cortex, which is independent of the well-described route through the apical micronemal/rhoptry complex. The pores may be the route by which motor proteins or other non micronemal surface proteins are trafficked, such as P25/P28 and SOAP, some of which are implicated in transmission blocking immunity.

P25 and P28 proteins of the malaria ookinete surface have multiple and partially redundant functions.

The ookinete surface proteins (P25 and P28) are proven antimalarial transmission-blocking vaccine targets, yet their biological functions are unknown. By using single (Sko) and double gene knock-out (Dko) Plasmodium berghei parasites, we show that P25 and P28 share multiple functions during ookinete/oocyst development. In the midgut of mosquitoes, the formation of ookinetes lacking both proteins (Dko parasites) is significantly inhibited due to decreased protection against lethal factors, including protease attack. In addition, Dko ookinetes have a much reduced capacity to traverse the midgut epithelium and to transform into the oocyst stage. P25 and P28 are partially redundant in these functions, since the efficiency of ookinete/oocyst development is only mildly compromised in parasites lacking either P25 or P28 (Sko parasites) compared with that of Dko parasites. The fact that Sko parasites are efficiently transmitted by the mosquito is a compelling reason for including both target antigens in transmission-blocking vaccines.