Target antigens in malaria transmission blocking immunity

Malaria transmission blocking immunity has been found to operate against two distinct phases of development of malaria parasites in the mosquito midgut: (i) against the extracellular gametes and newly fertilized zygotes shortly after ingestion by a mosquito of parasitized blood and (ii) against the zygotes during their subsequent development into ookinetes. Immunity is antibody-mediated and stage-specific. A set of three proteins, synthesized in the gametocytes, expressed on the surface of the gametes and newly fertilized zygotes and subsequently shed during later transformation of the zygotes, has been identified as the target antigens of anti-gamete fertilization blocking antibodies. A single protein, synthesized and expressed on the zygote surface during its development to ookinetes, has been identified as the target of antibodies which block the development of the fertilized parasites in the mosquito. Immunization of human populations against gamete or zygote antigens, while not directly protecting an immunized individual from inflection, would reduce the transfer of malaria within the population. Such immunity, in addition to reducing the overall rate of malaria transmission, would, if combined with a vaccine against the asexual (disease-causing) stages, reduce the chance of selection of parasites that are resistant to the asexual vaccine by preventing their entry into the mosquito population.     

Controlling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system

We are developing transgenic mosquitoes resistant to malaria parasites to test the hypothesis that genetically-engineered mosquitoes can be used to block the transmission of the parasites. We are developing and testing many of the necessary methodologies with the avian malaria parasite, Plasmodium gallinaceum, and its laboratory vector, Aedes aegypti, in anticipation of engaging the technical challenges presented by the malaria parasite, P. falciparum, and its major African vector, Anopheles gambiae. Transformation technology will be used to insert into the mosquito a synthetic gene for resistance to P. gallinaceum. The resistance gene will consist of a promoter of a mosquito gene controlling the expression of an effector protein that interferes with parasite development and/or infectivity. Mosquito genes whose promoter sequences are capable of sex- and tissue-specific expression of exogenous coding sequences have been identified, and stable transformation of the mosquito has been developed. We now are developing the expressed effector portion of the synthetic gene that will interfere with the transmission of the parasites. Mouse monoclonal antibodies that recognize the circumsporozoite protein of P. gallinaceum block sporozoite invasion of mosquito salivary glands, as well as abrogate the infectivity of sporozoites to a vertebrate host, the chicken, Gallus gallus, and block sporozoite invasion and development in susceptible cell lines in vitro. Using the genes encoding these antibodies, we propose to clone and express single-chain antibody constructs (scFv) that will serve as the effector portion of the gene that interferes with transmission of P. gallinaceum sporozoites.     

Mosquito-based transmission blocking vaccines for interrupting Plasmodium development

Reduction of transmission is critical for effective malaria control. Transmission blocking vaccines, which are intended to prevent the parasites from infecting the mosquito vectors, could target mosquito antigens that are required for the successful development of the parasite in its vector. Here we review recent advances in the identification of promising candidate antigens for a mosquito-based transmission blocking vaccine.     

Plasmodium-mosquito interactions,phage display libraries and transgenic mosquitoes impaired for malaria transmission

Malaria continues to kill millions of people every year and new strategies to combat this disease are urgently needed. Recent advances in the study of the mosquito vector and its interactions with the malaria parasite suggest that it may be possible to genetically manipulate the mosquito in order to reduce its vectorial capacity. Here we review the advances made to date in four areas: (1) the introduction of foreign genes into the mosquito germ line; (2) the characterization of tissue-specific promoters; (3) the identification of gene products that block development of the parasite in the mosquito; and (4) the generation of transgenic mosquitoes impaired for malaria transmission. While initial results show great promise, the problem of how to spread the blocking genes through wild mosquito populations remains to be solved.     

Heterologous expression of the C-terminal antigenic domain of the malaria vaccine candidate Pfs48/45 in the green algae Chlamydomonas reinhardtii

Malaria is a widespread and infectious disease that is a leading cause of death in many parts of the world. Eradication of malaria has been a major world health goal for decades, but one that still remains elusive. Other diseases have been eradicated using vaccination, but traditional vaccination methods have thus far been unsuccessful for malaria. Infection by Plasmodium species, the causative agent of malaria, is currently treated with drug-based therapies, but an increase in drug resistance has led to the need for new methods of treatment. A promising strategy for malaria treatment is to combine transmission blocking vaccines (TBVs) that prevent spread of disease with drug-based therapies to treat infected individuals. TBVs can be developed against surface protein antigens that are expressed during parasite reproduction in the mosquito. When the mosquito ingests blood from a vaccinated individual harboring the Plasmodium parasite, the antibodies generated by vaccination prevent completion of the parasites life-cycle. Animal studies have shown that immunization with Pfs48/45 results in the production of malaria transmission blocking antibodies; however, the development of this vaccine candidate has been hindered by poor expression in both prokaryotic and eukaryotic hosts. Recently, the chloroplast of Chlamydomonas reinhardtii has been used to express complex recombinant proteins. In this study, we show that the C-terminal antigenic region of the Pfs48/45 antigen can be expressed in the chloroplast of the green algae C. reinhardtii and that this recombinant protein has a conformation recognized by known transmission blocking antibodies. Production of this protein in algae has the potential to scale to the very large volumes required to meet the needs of millions at risk for contracting malaria.     

EGF domain II of protein Pb28 from Plasmodium berghei interacts with monoclonal transmission blocking antibody 13.1

Development of a vaccine against malaria is a major global health concern. The P28 proteins expressed on the surface of ookinetes of Plasmodium are the targets of transmission blocking antibodies. Injection of P28 proteins in vertebrate hosts induces antibodies that inhibit oocyst formation, blocking transmission of the parasite from mosquitos to human hosts. P28 proteins are crucial for parasite protection inside the mosquito midgut. Despite their importance, structural details of P28 family members have not been available to date. The purpose of this study was to structurally characterise a member of the P28 family, viz. Pb28 protein from Plasmodium berghei, and to study the interaction of Pb28 protein with the scFv (single chain variable fragment) of TBmAb (transmission blocking monoclonal antibody) 13.1 which blocks malaria transmission effectively. Pb28 protein and the TBmAb 13.1 scFv were modelled separately. To decipher the antigen–antibody interaction, ZDOCK and RDOCK programs were used. Our results suggest that, as compared to the template Pvs25, Pb28 protein has four EGF (epidermal growth factor)-like domains arranged in a triangular form with maximum root mean square deviations (RMSDs) present in the loop regions of EGF domains II and III. With the help of docking we were able to show that the B loop of EGF domain II of Pb28 protein interacts with the scFv of TBmAb 13.1. The predicted probable complex of Pb28 protein and 13.1 TBmAb suggests a mechanism for transmission blocking and may help in designing vaccine candidates in the absence of experimentally determined structures of these proteins. An erratum to this article can be found at     

Algae-produced Pfs25 elicits antibodies that inhibit malaria transmission

Subunit vaccines are significantly more expensive to produce than traditional vaccines because they are based primarily on recombinant proteins that must be purified from the expression system. Despite the increased cost, subunit vaccines are being developed because they are safe, effective, and can elicit antibodies that confer protection against diseases that are not currently vaccine-preventable. Algae are an attractive platform for producing subunit vaccines because they are relatively inexpensive to grow, genetically tractable, easily scaled to large volumes, have a short generation time, and are devoid of inflammatory, viral, or prion contaminants often present in other systems. We tested whether algal chloroplasts can produce malaria transmission blocking vaccine candidates, Plasmodium falciparum surface protein 25 (Pfs25) and 28 (Pfs28). Antibodies that recognize Pfs25 and Pfs28 disrupt the sexual development of parasites within the mosquito midgut, thus preventing transmission of malaria from one human host to the next. These proteins have been difficult to produce in traditional recombinant systems because they contain tandem repeats of structurally complex epidermal growth factor-like domains, which cannot be produced in bacterial systems, and because they are not glycosylated, so they must be modified for production in eukaryotic systems. Production in algal chloroplasts avoids these issues because chloroplasts can fold complex eukaryotic proteins and do not glycosylate proteins. Here we demonstrate that algae are the first recombinant system to successfully produce an unmodified and aglycosylated version of Pfs25 or Pfs28. These antigens are structurally similar to the native proteins and antibodies raised to these recombinant proteins recognize Pfs25 and Pfs28 from P. falciparum. Furthermore, antibodies to algae-produced Pfs25 bind the surface of in-vitro cultured P. falciparum sexual stage parasites and exhibit transmission blocking activity. Thus, algae are promising organisms for producing cysteine-disulfide-containing malaria transmission blocking vaccine candidate proteins.     

Plasmodium falciparum gametocytes: still many secrets of a hidden life

Sexual differentiation and parasite transmission are intimately linked in the life cycle of malaria parasites. The specialized cells providing this crucial link are the Plasmodium gametocytes. These are formed in the vertebrate host and are programmed to mature into gametes emerging from the erythrocytes in the midgut of a blood-feeding mosquito. The ensuing fusion into a zygote establishes parasite infection in the insect vector. Although key mechanisms of gametogenesis and fertilization are becoming progressively clear, the fundamental biology of gametocyte formation still presents open questions, some of which are specific to the human malaria parasite Plasmodium falciparum. Developmental commitment to sexual differentiation, regulation of stage-specific gene expression, the profound molecular and cellular changes accompanying gametocyte specialization, the requirement for tissue-specific sequestration in P. falciparum gametocytogenesis are proposed here as areas for future investigation. The epidemiological relevance of parasite transmission from humans to mosquito in the spread of malaria and of Plasmodium drug resistance genes indicates that understanding molecular mechanisms of gametocyte formation is highly relevant to design strategies able to interfere with the transmission of this disease.     

PfCCp proteins of Plasmodium falciparum: gametocyte-specific expression and role in complement-mediated inhibition of exflagellation

The sexual phase of the malaria parasite Plasmodium falciparum is essential for transmission of the disease and is accompanied by the co-ordinated expression of sexual stage proteins. Six of these proteins belong to a highly conserved apicomplexan family of multi-domain adhesion proteins, termed PfCCps. PfCCp1, PfCCp2 and PfCCp3 are co-dependently expressed in the parasitophorous vacuole associated with the gametocyte plasma membrane. PfCCp2 and PfCCp3 also play an essential role for parasite development in the mosquito. We show that the six PfCCp proteins are expressed in stages II-V of gametocytogenesis as well as during early gamete formation. The proteins are expressed in association with the surface of both male and female gametocytes and macrogametes, but are not present in exflagellating microgametes. Further, the newly described protein PfCCp4 co-localizes with the transmission blocking candidate Pfs230, with which it forms a protein complex. In contrast to the phenotypes that are observed following targeted gene disruption of PfCCp2, PfCCp3 or Pfs230, the lack of PfCCp4 expression does not inhibit parasite development in the mosquito vector. This indicates a non-essential role for this protein during parasite transmission. Exflagellation assays revealed that antibodies directed against distinct domains of PfCCp1 through PfCCp4 and PfFNPA support a complement-mediated decrease in gametocyte emergence. We conclude that the six PfCCp proteins are specifically expressed during gametocytogenesis and gamete formation, and that select members may represent prospective candidates for transmission blocking vaccines.     

Human immune responses against sexual stages of malaria parasites: considerations for malaria vaccines

Studies on the natural immune responses to the sexual stages of malaria parasites have been reviewed in the context of human malaria transmission-blocking vaccines. Antibodies against the sexual stages of the malaria parasite, gametocytes and gametes, are readily evoked by natural malaria infections. These antibodies that suppress infectivity at high concentrations can, at low concentrations, enhance the development of the parasite in the mosquito; however, because enhancing antibodies are prevalent during natural malaria infections, it is likely that a vaccine would rapidly boost these antibodies to blocking levels. The immunogenicity of sexual stage antigens appears to be constrained in the human host, probably due to T epitope polymorphism and MHC restriction in humans. These constraints apply mainly to those antigens that are sensitive targets of host immunity such as the gamete surface antigens and not to internal gamete antigens, indicating that antigenic polymorphism may have evolved in response to immune selection pressure. Evidence for immunosuppression of the host by exposure to endemic malaria is presented and its consequences on vaccine development are discussed.     

The effects of mosquito transmission and population bottlenecking on virulence, multiplication rate and rosetting in rodent malaria

Malaria parasites vary in virulence, but the effects of mosquito transmission on virulence phenotypes have not been systematically analysed. Using six lines of malaria parasite that varied widely in virulence, three of which had been serially blood-stage passaged many times, we found that mosquito transmission led to a general reduction in malaria virulence. Despite that, the between-line variation in virulence remained. Forcing serially passaged lines through extreme population bottlenecks (<5 parasites) reduced virulence in only one of two lines. That reduction was to a level intermediate between that of the virulent parental and avirulent ancestral line. Mosquito transmission did not reverse the increased parasite replication rates that had accrued during serial passage, but it did increase rosetting frequencies. Re-setting of asexual stage genes during the sexual stages of the life cycle, coupled with stochastic sampling of parasites with variable virulence during population bottlenecks, could account for the virulence reductions and increased rosetting induced by mosquito transmission.     

Blocking of malaria parasite development in mosquito and fecundity reduction by midgut antibodies in Anopheles stephensi (Diptera: Culicidae)

Rabbits were immunized three times with extracts of Anopheles stephensi midgut. Immunized rabbits showed a high titer of antibodies when characterized by ELISA. We investigated the effect of anti-mosquito midgut antibodies on mosquito fecundity, longevity, mortality, engorgement, and the development of the malaria parasite in mosquitoes. Fecundity was reduced significantly (38%) and similarly hatchability by about 43.5%. There was no statistically significant effect on mortality, longevity, and engorgement. When the mosquito blood meal contained anti-midgut antibodies, fewer oocysts of Plasmodium vivax developed in the mosquito midgut and the proportion of mosquitoes becoming infected was significantly reduced. We also found that the midgut antibodies inhibit the development and/or translocation of the sporozoites. Antisera raised against midgut of A. stephensi recognized eight polypeptides (110, 92, 70, 45, 38, 29, 15, 13 kDa) by Western blotting. Cross-reactive antigens/epitopes present in other tissues of A. stephensi were also examined both by Western blotting and in vivo ELISA. Together, these observations open an avenue for research toward the development of a vector-based malaria parasite transmission blocking vaccine and/or anti-mosquito vaccine.     

The multifaceted mosquito anti-Plasmodium response

Plasmodium development within its mosquito vector is an essential step in malaria transmission, as illustrated in world regions where malaria was successfully eradicated via vector control. The innate immune system of most mosquitoes is able to completely clear a Plasmodium infection, preventing parasite transmission to humans. Understanding the biological basis of this phenomenon is expected to inspire new strategies to curb malaria incidence in countries where vector control via insecticides is unpractical, or inefficient because insecticide resistance genes have spread across mosquito populations. Several aspects of mosquito biology that condition the success of the parasite in colonizing its vector begin to be understood at the molecular level, and a wealth of recently published data highlights the multifaceted nature of the mosquito response against parasite invasion. In this brief review, we attempt to provide an integrated view of the challenges faced by the parasite to successfully invade its mosquito host, and discuss the possible intervention strategies that could exploit this knowledge for the fight against human malaria.     

Inference and dynamic simulation of malaria using a simple climate-driven entomological model of malaria transmission

Given the crucial role of climate in malaria transmission, many mechanistic models of malaria represent vector biology and the parasite lifecycle as functions of climate variables in order to accurately capture malaria transmission dynamics. Lower dimension mechanistic models that utilize implicit vector dynamics have relied on indirect climate modulation of transmission processes, which compromises investigation of the ecological role played by climate in malaria transmission. In this study, we develop an implicit process-based malaria model with direct climate-mediated modulation of transmission pressure borne through the Entomological Inoculation Rate (EIR). The EIR, a measure of the number of infectious bites per person per unit time, includes the effects of vector dynamics, resulting from mosquito development, survivorship, feeding activity and parasite development, all of which are moderated by climate. We combine this EIR-model framework, which is driven by rainfall and temperature, with Bayesian inference methods, and evaluate the model’s ability to simulate local transmission across 42 regions in Rwanda over four years. Our findings indicate that the biologically-motivated, EIR-model framework is capable of accurately simulating seasonal malaria dynamics and capturing of some of the inter-annual variation in malaria incidence. However, the model unsurprisingly failed to reproduce large declines in malaria transmission during 2018 and 2019 due to elevated anti-malaria measures, which were not accounted for in the model structure. The climate-driven transmission model also captured regional variation in malaria incidence across Rwanda’s diverse climate, while identifying key entomological and epidemiological parameters important to seasonal malaria dynamics. In general, this new model construct advances the capabilities of implicitly-forced lower dimension dynamical malaria models by leveraging climate drivers of malaria ecology and transmission.     

A model for the coevolution of immunity and immune evasion in vector-borne diseases with implications for the epidemiology of malaria

We describe a model of host-parasite coevolution, where the interaction depends on the investments by the host in its immune response and by the parasite in its ability to suppress (or evade) its host's immune response. We base our model on the interaction between malaria parasites and their mosquito hosts and thus describe the epidemiological dynamics with the Macdonald-Ross equation of malaria epidemiology. The qualitative predictions of the model are most sensitive to the cost of the immune response and to the intensity of transmission. If transmission is weak or the cost of immunity is low, the system evolves to a coevolutionarily stable equilibrium at intermediate levels of investment (and, generally, at a low frequency of resistance). At a higher cost of immunity and as transmission intensifies, the system is not evolutionarily stable but rather cycles around intermediate levels of investment. At more intense transmission, neither host nor parasite invests any resources in dominating its partner so that no resistance is observed in the population. These results may help to explain the lack of encapsulated malaria parasites generally observed in natural populations of mosquito vectors, despite strong selection pressure for resistance in areas of very intense transmission.     

The Plasmodium bottleneck: malaria parasite losses in the mosquito vector

Nearly one million people are killed every year by the malaria parasite Plasmodium. Although the disease-causing forms of the parasite exist only in the human blood, mosquitoes of the genus Anopheles are the obligate vector for transmission. Here, we review the parasite life cycle in the vector and highlight the human and mosquito contributions that limit malaria parasite development in the mosquito host. We address parasite killing in its mosquito host and bottlenecks in parasite numbers that might guide intervention strategies to prevent transmission.     

Synthetic propeptide inhibits mosquito midgut chitinase and blocks sporogonic development of malaria parasite

Incessant transmission of the parasite by mosquitoes makes most attempts to control malaria fail. Blocking of parasite transmission by mosquitoes therefore is a rational strategy to combat the disease. Upon ingestion of blood meal mosquitoes secrete chitinase into the midgut. This mosquito chitinase is a zymogen which is activated by the removal of a propeptide from the N-terminal. Since the midgut peritrophic matrix acts as a physical barrier, the activated chitinase is likely to contribute to the further development of the malaria parasite in the mosquito. Earlier it has been shown that inhibiting chitinase activity in the mosquito midgut blocked sporogonic development of the malaria parasite. Since synthetic propeptides of several zymogens have been found to be potent inhibitors of their respective enzymes, we tested propeptide of mosquito midgut chitinase as an inhibitor and found that the propeptide almost completely inhibited the recombinant or purified native Anopheles gambiae chitinase. We also examined the effect of the inhibitory peptide on malaria parasite development. The result showed that the synthetic propeptide blocked the development of human malaria parasite Plasmodium falciparum in the African malaria vector An. gambiae and avian malaria parasite Plasmodium gallinaceum in Aedes aegypti mosquitoes. This study implies that the expression of inhibitory mosquito midgut chitinase propeptide in response to blood meal may alter the mosquito's vectorial capacity. This may lead to developing novel strategies for controlling the spread of malaria.     

Last-come,best served? Mosquito biting order and Plasmodium transmission

A pervasive characteristic of parasite infections is their tendency to be overdispersed. Understanding the mechanisms underlying this overdispersed distribution is of key importance as it may impact the transmission dynamics of the pathogen. Although multiple factors ranging from environmental stochasticity to inter-individual heterogeneity may explain parasite overdispersion, parasite infection is also overdispersed in an inbred host population maintained under laboratory conditions, suggesting that other mechanisms are at play. Here, we show that the aggregated distribution of malaria parasites within mosquito vectors is partially explained by a temporal heterogeneity in parasite infectivity triggered by the bites of mosquitoes. Parasite transmission tripled between the mosquito''s first and last blood feed in a period of only 3 h. Surprisingly, the increase in transmission is not associated with an increase in parasite investment in production of the transmissible stage. Overall, we highlight that Plasmodium is capable of responding to the bites of mosquitoes to increase its own transmission at a much faster pace than initially thought and that this is partly responsible for overdispersed distribution of infection. We discuss the underlying mechanisms as well as the broader implications of this plastic response for the epidemiology of malaria.     

Transgenic mosquitoes and malaria transmission

As the malaria burden persists in most parts of the developing world, the concept of implementation of new strategies such as the use of genetically modified mosquitoes to control the disease continues to gain support. In Africa, which suffers most from malaria, mosquito vector populations are spread almost throughout the entire continent, and the parasite reservoir is big and continuously increasing. Moreover, malaria is transmitted by many species of anophelines with specific seasonal and geographical patterns. Therefore, a well designed, evolutionarily robust and publicly accepted plan aiming at population reduction or replacement is required. The task is twofold: to engineer mosquitoes with a genetic trait that confers resistance to malaria or causes population suppression; and, to drive the new trait through field populations. This review examines these two issues, and describes the groundwork that has been done towards understanding of the complex relation between the parasite and its vector.     

Plasmodium vivax gametocytes and transmission

Malaria elimination means cessation of parasite transmission. At present, the declining malaria incidence in many countries has made elimination a feasible goal. Transmission control has thus been placed at the center of the national malaria control programs. The efficient transmission of Plasmodium vivax from humans to mosquitoes is a key factor that helps perpetuate malaria in endemic areas. A better understanding of transmission is crucial to the success of elimination efforts. Biological delineation of the parasite transmission process is important for identifying and prioritizing new targets of intervention. Identification of the infectious parasite reservoir in the community is key to devising an effective elimination strategy. Here we describe the fundamental characteristics of P. vivax gametocytes - the dynamics of their production, longevity, and the relationship with the total parasitemia - as well as recent advances in the molecular understanding of parasite sexual development. In relation to malaria elimination, factors influencing the human infectivity and the current evidence for a role of asymptomatic carriers in transmission are presented.