Injection Of Sporozoites Research Articles

Experimental vaccination by single dose sporozoite injection of blood-stage attenuated malaria parasites

Malaria vaccination approaches using live Plasmodium parasites are currently explored, with either attenuated mosquito-derived sporozoites or attenuated blood-stage parasites. Both approaches would profit from the availability of attenuated and avirulent parasites with a reduced blood-stage multiplication rate. Here we screened gene-deletion mutants of the rodent parasite P. berghei and the human parasite P. falciparum for slow growth. Furthermore, we tested the P. berghei mutants for avirulence and resolving blood-stage infections, while preserving sporozoite formation and liver infection. Targeting 51 genes yielded 18 P. berghei gene-deletion mutants with several mutants causing mild infections. Infections with the two most attenuated mutants either by blood stages or by sporozoites were cleared by the immune response. Immunization of mice led to protection from disease after challenge with wild-type sporozoites. Two of six generated P. falciparum gene-deletion mutants showed a slow growth rate. Slow-growing, avirulent P. falciparum mutants will constitute valuable tools to inform on the induction of immune responses and will aid in developing new as well as safeguarding existing attenuated parasite vaccines.

Innate immune activation restricts priming and protective efficacy of the radiation-attenuated PfSPZ malaria vaccine.

A systems analysis was conducted to determine the potential molecular mechanisms underlying differential immunogenicity and protective efficacy results of a clinical trial of the radiation-attenuated whole-sporozoite PfSPZ vaccine in African infants. Innate immune activation and myeloid signatures at prevaccination baseline correlated with protection from P. falciparum parasitemia in placebo controls. These same signatures were associated with susceptibility to parasitemia among infants who received the highest and most protective PfSPZ vaccine dose. Machine learning identified spliceosome, proteosome, and resting DC signatures as prevaccination features predictive of protection after highest-dose PfSPZ vaccination, whereas baseline circumsporozoite protein-specific (CSP-specific) IgG predicted nonprotection. Prevaccination innate inflammatory and myeloid signatures were associated with higher sporozoite-specific IgG Ab response but undetectable PfSPZ-specific CD8+ T cell responses after vaccination. Consistent with these human data, innate stimulation in vivo conferred protection against infection by sporozoite injection in malaria-naive mice while diminishing the CD8+ T cell response to radiation-attenuated sporozoites. These data suggest a dichotomous role of innate stimulation for malaria protection and induction of protective immunity by whole-sporozoite malaria vaccines. The uncoupling of vaccine-induced protective immunity achieved by Abs from more protective CD8+ T cell responses suggests that PfSPZ vaccine efficacy in malaria-endemic settings may be constrained by opposing antigen presentation pathways.

Liver-Directed AAV8 Booster Vaccine Expressing Plasmodium falciparum Antigen Following Adenovirus Vaccine Priming Elicits Sterile Protection in a Murine Model.

Hepatocyte infection by malaria sporozoites is a bottleneck in the life-cycle of Plasmodium spp. including P. falciparum, which causes the most lethal form of malaria. Therefore, developing an effective vaccine capable of inducing the strong humoral and cellular immune responses necessary to block the pre-erythrocytic stage has potential to overcome the spatiotemporal hindrances pertaining to parasite biology and hepatic microanatomy. We recently showed that when combined with a human adenovirus type 5 (AdHu5)-priming vaccine, adeno-associated virus serotype 1 (AAV1) is a potent booster malaria vaccine vector capable of inducing strong and long-lasting protective immune responses in a rodent malaria model. Here, we evaluated the protective efficacy of a hepatotropic virus, adeno-associated virus serotype 8 (AAV8), as a booster vector because it can deliver a transgene potently and rapidly to the liver, the organ malaria sporozoites initially infect and multiply in following sporozoite injection by the bite of an infected mosquito. We first generated an AAV8-vectored vaccine expressing P. falciparum circumsporozoite protein (PfCSP). Intravenous (i.v.) administration of AAV8-PfCSP to mice initially primed with AdHu5-PfCSP resulted in a hepatocyte transduction rate ~2.5 times above that seen with intramuscular (i.m.) administration. This immunization regimen provided a better protection rate (100% sterile protection) than that of the i.m. AdHu5-prime/i.m. AAV8-boost regimen (60%, p < 0.05), i.m. AdHu5-prime/i.v. AAV1-boost (78%), or i.m. AdHu5-prime/i.m. AAV1-boost (80%) against challenge with transgenic PfCSP-expressing P. berghei sporozoites. Compared with the i.m. AdHu5-prime/i.v. AAV1-boost regimen, three other regimens induced higher levels of PfCSP-specific humoral immune responses. Importantly, a single i.v. dose of AAV8-PfCSP recruited CD8+ T cells, especially resident memory CD8+ T cells, in the liver. These data suggest that boost with i.v. AAV8-PfCSP can improve humoral and cellular immune responses in BALB/c mice. Therefore, this regimen holds great promise as a next-generation platform for the development of an effective malaria vaccine.

Comparative intravital imaging of human and rodent malaria sporozoites reveals the skin is not a species‐specific barrier

Malaria infection starts with the injection of Plasmodium sporozoites into the host’s skin. Sporozoites are motile and move in the skin to find and enter blood vessels to be carried to the liver. Here, we present the first characterization of P. falciparum sporozoites in vivo, analyzing their motility in mouse skin and human skin xenografts and comparing their motility to two rodent malaria species. These data suggest that in contrast to the liver and blood stages, the skin is not a species‐specific barrier for Plasmodium. Indeed, P. falciparum sporozoites enter blood vessels in mouse skin at similar rates to the rodent malaria parasites. Furthermore, we demonstrate that antibodies targeting sporozoites significantly impact the motility of P. falciparum sporozoites in mouse skin. Though the sporozoite stage is a validated vaccine target, vaccine trials have been hampered by the lack of good animal models for human malaria parasites. Pre‐clinical screening of next‐generation vaccines would be significantly aided by the in vivo platform we describe here, expediting down‐selection of candidates prior to human vaccine trials.

Role of Dual Oxidase 1 in severe malaria

Abstract Despite important advances in malaria control, the disease is still a major public health issue in sub-Saharan Africa. Recent reports indicate over 400,000 deaths annually. Parasite resistance to known antimalarial drugs delays control and elimination. Dual oxidase 1 (Duox1), a member of the NADPH oxidase enzyme family, generates hydrogen peroxide that is used by its partner enzyme lactoperoxidase to generate antimicrobial hypothiocyanite. Malaria parasites are susceptible to hypothiocyanite in vitro. Duox1 is required for the generation of hypothiocyanite in vivo. We hypothesized that malaria parasites are susceptible to the Duox1-mediated antimicrobial system in vivo. Duox1-deficient and wild-type, Duox1-expressing mice were infected with Plasmodium berghei by either intravenous injection of sporozoites or exposure to infected mosquitos. The results indicate that Duox1-deficient mice either infected with intravenous injection of P. berghei sporozoites (p&amp;lt;0.004) or by exposure to P. berghei-infected mosquito bites (p&amp;lt;0.002), have significantly higher blood parasite density at peak parasitemia compared to wild-type mice. Survival data indicated that Duox1-deficient mice are more susceptible to P. berghei infection than control animals (p&amp;lt;0.03 for intravenous infection, p&amp;lt;0.02 for exposure to mosquito bites). Histopathological analysis of brain tissues revealed a slightly higher number of indices of cerebral pathology including cerebrovascular infected red blood cells and hemozoin accumulation, hemostasis, and hemorrhage. Overall, these data propose a novel role of Duox1 in delaying the onset of parasitemia and clinical outcomes in malaria infection.

Plasmodium vivax Cell Traversal Protein for Ookinetes and Sporozoites (CelTOS) Functionally Restricted Regions Are Involved in Specific Host-Pathogen Interactions.

Following the injection of Plasmodium sporozoites by a female Anopheles mosquito into the dermis, they become engaged on a long journey to hepatic tissue where they must migrate through different types of cell to become established in parasitophorous vacuoles in hepatocytes. Studies have shown that proteins such as cell traversal protein for Plasmodium ookinetes and sporozoites (CelTOS) play a crucial role in cell-traversal ability. Although CelTOS has been extensively studied in various species and included in pre-clinical assays it remains unknown which P. vivax CelTOS (PvCelTOS) regions are key in its interaction with traversed or target cells (Kupffer or hepatocytes) and what type of pressure, association and polymorphism these important regions could have to improve their candidacy as important vaccine antigens. This work has described producing a recombinant PvCelTOS which was recognized by ~30% P. vivax-infected individuals, thereby confirming its ability for inducing a natural immune response. PvCelTOS' genetic diversity in Colombia and its ability to interact with HeLa (traversal cell) and/or HepG2 cell (target cell) external membrane have been assessed. One region in the PvCelTOS amino-terminal region and another in its C-terminus were seen to be participating in host-pathogen interactions. These regions had important functional constraint signals (ω < 0.3 and several sites under negative selection) and were able to inhibit specific rPvCelTOS binding to HeLa cells. This led to suggesting that sequences between aa 41–60 (40833) and 141–160 (40838) represent promising candidates for an anti-P. vivax subunit-based vaccine.

Activated Plasmodium-specific CD8 T cells find liver stages mostly randomly

Abstract Malaria is a major infectious disease, with 500,000 people dying every year. Malaria infection starts when an infected mosquito injects malaria sporozoites during feeding which migrate to the liver and form liver stages. Elimination of liver stages is a major feature of CD8 T cell-based malaria vaccines. It is established that vaccines inducing high numbers of memory CD8 T cells are capable of eliminating all liver stages, preventing clinical malaria. However, how CD8 T cells are capable of doing that in the 48-hour liver stage in mice remains unclear. One possibility is that as sporozoites invade hepatocytes from the blood, they induce local inflammation, acting as an attraction signal for T cells. We performed experiments in which fluorescent-labeled Plasmodium sporozoites and Plasmodium-specific and non-specific CD8 T cells were tracked in mouse livers using intravital microscopy. To analyze the data we used three metrics to measure attraction of T cells to the infection site based either on angles or distances from a T cell to the parasite. We found that for the majority of imaged T cells, there is no evidence of attraction to the parasite, indicating that in this experimental system, vaccine-induced CD8 T cells locate the infection site randomly. By modeling T cells searching for the infection site and utilizing experimentally measured speeds of T cell movement in the liver, we found that if enough memory CD8 T cells are present in the liver, all parasites can be found within 48 hours after sporozoite injection. Taken together, our results suggest that memory CD8 T cells in the liver perform the search for infection randomly, precluding the need to find specific signals and receptors of T cell attraction to the infection site.

Down-selecting circumsporozoite protein-based malaria vaccine: A comparison of malaria sporozoite challenge model.

Plasmodium falciparum circumsporozoite protein (PfCSP) is the main target antigen in development of pre-erythrocytic malaria vaccines. To evaluate PfCSP vaccines in animal models, challenge by intravenous sporozoite injection is preferentially used. However, in clinical trials, vaccinated human volunteers are exposed to the bites of malaria-infected mosquitoes. In this study, we down-selected Escherichia coli-produced full-length PfCSP (PfCSP-F) and its three truncated PfCSPs based on their abilities to elicit immune response and protection in mice against two challenge models. We showed that immunization with three doses of PfCSP-F elicited high anti-PfCSP antibody titres and 100% protection against the bites of infected mosquitoes. Meanwhile, three-dose truncated PfCSP induced 60%-70% protection after immunization with each truncated PfCSP. Heterologous prime-boost immunization regimen with adenovirus-PfCSP-F and R32LR greatly induced complete protection against intravenous sporozoite injection. Our results suggest that Abs to both anti-repeat and anti-nonrepeat regions induced by PfCSP-F are required to confer complete protection against challenge by the bites of infected mosquitoes, whereas anti-repeat Abs play an important role in protection against intravenous sporozoite injection. Our findings provide a potential clinical application that PfCSP-F vaccine induces potent Abs capable of neutralizing sporozoites in the dermis inoculated by infected mosquitoes and subsequently sporozoites in the blood circulation.

Antibody-Mediated Protection against Plasmodium Sporozoites Begins at the Dermal Inoculation Site.

Plasmodium sporozoites are injected into the skin as mosquitoes probe for blood. From here, they migrate through the dermis to find blood vessels which they enter in order to be rapidly carried to the liver, where they invade hepatocytes and develop into the next life cycle stage, the exoerythrocytic stage. Once sporozoites enter the blood circulation, they are found in hepatocytes within minutes. In contrast, sporozoite exit from the inoculation site resembles a slow trickle and occurs over several hours. Thus, sporozoites spend the majority of their extracellular time at the inoculation site, raising the hypothesis that this is when the malarial parasite is most vulnerable to antibody-mediated destruction. Here, we investigate this hypothesis and demonstrate that the neutralizing capacity of circulating antibodies is greater at the inoculation site than in the blood circulation. Furthermore, these antibodies are working, at least in part, by impacting sporozoite motility at the inoculation site. Using actively and passively immunized mice, we found that most parasites are either immobilized at the site of injection or display reduced motility, particularly in their net displacement. We also found that antibodies severely impair the entry of sporozoites into the bloodstream. Overall, our data suggest that antibodies targeting the migratory sporozoite exert a large proportion of their protective effect at the inoculation site.IMPORTANCE Studies in experimental animal models and humans have shown that antibodies against Plasmodium sporozoites abolish parasite infectivity and provide sterile immunity. While it is well documented that these antibodies can be induced after immunization with attenuated parasites or subunit vaccines, the mechanisms by and location in which they neutralize parasites have not been fully elucidated. Here, we report studies indicating that these antibodies display a significant portion of their protective effect in the skin after injection of sporozoites and that one mechanism by which they work is by impairing sporozoite motility, thus diminishing their ability to reach blood vessels. These results suggest that immune protection against malaria begins at the earliest stages of parasite infection and emphasize the need of performing parasite challenge in the skin for the evaluation of protective immunity.

Immunization efficacy of cryopreserved genetically attenuated Plasmodium berghei sporozoites.

Malaria is transmitted through the injection of Plasmodium sporozoites into the skin by Anopheles mosquitoes. The parasites first replicate within the liver before infecting red blood cells, which leads to the symptoms of the disease. Experimental immunization with attenuated sporozoites that arrest their development in the liver has been extensively investigatedin rodent models and humans. Recent technological advances have included the capacity to cryopreserve sporozoites for injection, which has enabled a series of controlled studies on human infection with sporozoites. Here, we used a cryopreservation protocol to test the efficiency of genetically attenuated cryopreserved sporozoites for immunization of mice in comparison with freshly isolated controls. This showed that cryopreserved sporozoites are highly viable as judged by their capacity to migrate in vitro but show only 20% efficiency in liver infection, which impacts their capacity to generate protection of animals in immunization experiments.

Insights into the early liver stage biology of Plasmodium.

Even though malaria is preventable and curable, it has become a serious threat to mankind. In 2016, there were an estimated 216 million cases of malaria across the world. The biology of its causative agent, i.e. Plasmodium parasite is full of complex mechanisms. There are five Plasmodium species responsible for malaria in humans, viz. Plasmodium falciparum, P. vivax, P. malariae, P. ovale and recently identified P. knowlesi that normally infect apes. In humans, malaria is spread by the injection of Plasmodium sporozoites through the bite of infectious Anopheles' female mosquito during their blood meal. From the time of entry into human skin till the development into the asexual forms, the parasite undergoes several transformations. This review attempts to understand the science behind the pre-erythrocytic liver stage of Plasmodium. Research articles explaining parasite biology, cell-traversal, transformation stages, cell-egress process, etc. were retrieved from PubMed and google scholar database. Various known and unknown mechanisms and strategies used by the malaria parasite P. berghei in rodent models have been discussed in this review. Limited or no information was available for humans, due to technical feasibility and complexity of parasite's life cycle. Hence, it was concluded that there is an urgent need to investigate the hepatic invasion, traversal and egress mechanism of P. falciparum and P. vivax for developing novel therapeutics to fight against malaria.

Controlled Human Malaria Infection: Applications, Advances, and Challenges.

Controlled human malaria infection (CHMI) entails deliberate infection with malaria parasites either by mosquito bite or by direct injection of sporozoites or parasitized erythrocytes. When required, the resulting blood-stage infection is curtailed by the administration of antimalarial drugs. Inducing a malaria infection via inoculation with infected blood was first used as a treatment (malariotherapy) for neurosyphilis in Europe and the United States in the early 1900s. More recently, CHMI has been applied to the fields of malaria vaccine and drug development, where it is used to evaluate products in well-controlled early-phase proof-of-concept clinical studies, thus facilitating progression of only the most promising candidates for further evaluation in areas where malaria is endemic. Controlled infections have also been used to immunize against malaria infection. Historically, CHMI studies have been restricted by the need for access to insectaries housing infected mosquitoes or suitable malaria-infected individuals. Evaluation of vaccine and drug candidates has been constrained in these studies by the availability of a limited number of Plasmodium falciparum isolates. Recent advances have included cryopreservation of sporozoites, the manufacture of well-characterized and genetically distinct cultured malaria cell banks for blood-stage infection, and the availability of Plasmodium vivax-specific reagents. These advances will help to accelerate malaria vaccine and drug development by making the reagents for CHMI more widely accessible and also enabling a more rigorous evaluation with multiple parasite strains and species. Here we discuss the different applications of CHMI, recent advances in the use of CHMI, and ongoing challenges for consideration.

The Threshold of Protection from Liver-Stage Malaria Relies on a Fine Balance between the Number of Infected Hepatocytes and Effector CD8+ T Cells Present in the Liver.

Since the demonstration of sterile protection afforded by injection of irradiated sporozoites, CD8+ T cells have been shown to play a significant role in protection from liver-stage malaria. This is, however, dependent on the presence of an extremely high number of circulating effector cells, thought to be necessary to scan, locate, and kill infected hepatocytes in the short time that parasites are present in the liver. We used an adoptive transfer model to elucidate the kinetics of the effector CD8+ T cell response in the liver following Plasmodium berghei sporozoite challenge. Although effector CD8+ T cells require <24 h to find, locate, and kill infected hepatocytes, active migration of Ag-specific CD8+ T cells into the liver was not observed during the 2-d liver stage of infection, as divided cells were only detected from day 3 postchallenge. However, the percentage of donor cells recruited into division was shown to indicate the level of Ag presentation from infected hepatocytes. By titrating the number of transferred Ag-specific effector CD8+ T cells and sporozoites, we demonstrate that achieving protection toward liver-stage malaria is reliant on CD8+ T cells being able to locate infected hepatocytes, resulting in a protection threshold dependent on a fine balance between the number of infected hepatocytes and CD8+ T cells present in the liver. With such a fine balance determining protection, achieving a high number of CD8+ T cells will be critical to the success of a cell-mediated vaccine against liver-stage malaria.

Efficient monitoring of the blood-stage infection in a malaria rodent model by the rotating-crystal magneto-optical method.

Intense research efforts have been focused on the improvement of the efficiency and sensitivity of malaria diagnostics, especially in resource-limited settings for the detection of asymptomatic infections. Our recently developed magneto-optical (MO) method allows the accurate quantification of malaria pigment crystals (hemozoin) in blood by their magnetically induced rotation. First evaluations of the method using β-hematin crystals and in vitro P. falciparum cultures implied its potential for high-sensitivity malaria diagnosis. To further investigate this potential, here we study the performance of the method in monitoring the in vivo onset and progression of the blood-stage infection in a rodent malaria model. Our results show that the MO method can detect the first generation of intraerythrocytic P. berghei parasites 66–76 hours after sporozoite injection, demonstrating similar sensitivity to Giesma-stained light microscopy and exceeding that of flow cytometric techniques. Magneto-optical measurements performed during and after the treatment of P. berghei infections revealed that both the follow up under treatment and the detection of later reinfections are feasible with this new technique. The present study demonstrates that the MO method – besides being label and reagent-free, automated and rapid – has a high in vivo sensitivity and is ready for in-field evaluation.

Longitudinal analysis of Plasmodium sporozoite motility in the dermis reveals component of blood vessel recognition.

Malaria infection starts with injection of Plasmodium sporozoites by an Anopheles mosquito into the skin of the mammalian host. How sporozoites locate and enter a blood vessel is a critical, but poorly understood process. In this study, we examine sporozoite motility and their interaction with dermal blood vessels, using intravital microscopy in mice. Our data suggest that sporozoites exhibit two types of motility: in regions far from blood vessels, they exhibit 'avascular motility', defined by high speed and less confinement, while in the vicinity of blood vessels their motility is more constrained. We find that curvature of sporozoite tracks engaging with vasculature optimizes contact with dermal capillaries. Imaging of sporozoites with mutations in key adhesive proteins highlight the importance of the sporozoite's gliding speed and its ability to modulate adhesive properties for successful exit from the inoculation site.

Assessment of Parasite Liver-Stage Burden in Human-Liver Chimeric Mice.

Humanized mice with a chimeric liver are a promising tool to evaluate the "in vivo" efficacy of novel compounds or vaccine-induced antibodies directed against the pre-erythrocytic stages of Plasmodium falciparum. The absence of human red blood cells in these humanized mice precludes the transition from liver to blood stage. The qPCR-based method described below allows for a sensitive and reliable quantification of parasite DNA in the chimeric liver following a challenge via infected mosquito bite or intravenous injection of sporozoites. With this method approximately 25 % of the total chimeric liver is examined and a single infected hepatocyte can be detected in the analyzed tissue. The use of appropriate species-specific probes can also allow for the detection of other Plasmodium species in vivo.

Protection from liver-stage malaria is dependent on a fine balance between the number of infected hepatocytes and effector CD8+ T cells

Background Since the demonstration of sterile protection afforded by injection of irradiated sporozoites, CD8 T cells have been shown to play a significant role in protection from liver-stage malaria. This is, however, dependent on the presence of an extremely high number of circulating effector cells, thought to be necessary to scan, locate and kill infected hepatocytes in the short time that parasites are present in the liver. To this day very little is known about the T cell response to malaria in the liver, therefore we have utilized an adoptive transfer system in mice to elucidate the kinetics of CD8 T cell mediated protection following sporozoite challenge.

Local immune response to injection of Plasmodium sporozoites into the skin.

Malarial infection is initiated when the sporozoite form of the Plasmodium parasite is inoculated into the skin by a mosquito. Sporozoites invade hepatocytes in the liver and develop into the erythrocyte-infecting form of the parasite, the cause of clinical blood infection. Protection against parasite development in the liver can be induced by injection of live attenuated parasites that do not develop in the liver and thus do not cause blood infection. Radiation-attenuated sporozoites (RAS) and genetically attenuated parasites are now considered as lead candidates for vaccination of humans against malaria. Although the skin appears as the preferable administration route, most studies in rodents, which have served as model systems, have been performed after i.v. injection of attenuated sporozoites. In this study, we analyzed the early response to Plasmodium berghei RAS or wild-type sporozoites (WTS) injected intradermally into C57BL/6 mice. We show that RAS have a similar in vivo distribution to WTS and that both induce a similar inflammatory response consisting of a biphasic recruitment of polymorphonuclear neutrophils and inflammatory monocytes in the skin injection site and proximal draining lymph node (dLN). Both WTS and RAS associate with neutrophils and resident myeloid cells in the skin and the dLN, transform inside CD11b(+) cells, and induce a Th1 cytokine profile in the dLN. WTS and RAS are also similarly capable of priming parasite-specific CD8(+) T cells. These studies delineate the early and local response to sporozoite injection into the skin, and suggest that WTS and RAS prime the host immune system in a similar fashion.

Using infective mosquitoes to challenge monkeys with Plasmodium knowlesi in malaria vaccine studies.

BackgroundWhen rhesus monkeys (Macaca mulatta) are used to test malaria vaccines, animals are often challenged by the intravenous injection of sporozoites. However, natural exposure to malaria comes via mosquito bite, and antibodies can neutralize sporozoites as they traverse the skin. Thus, intravenous injection may not fairly assess humoral immunity from anti-sporozoite malaria vaccines. To better assess malaria vaccines in rhesus, a method to challenge large numbers of monkeys by mosquito bite was developed.MethodsSeveral species and strains of mosquitoes were tested for their ability to produce Plasmodium knowlesi sporozoites. Donor monkey parasitaemia effects on oocyst and sporozoite numbers and mosquito mortality were documented. Methylparaben added to mosquito feed was tested to improve mosquito survival. To determine the number of bites needed to infect a monkey, animals were exposed to various numbers of P. knowlesi-infected mosquitoes. Finally, P. knowlesi-infected mosquitoes were used to challenge 17 monkeys in a malaria vaccine trial, and the effect of number of infectious bites on monkey parasitaemia was documented.ResultsAnopheles dirus, Anopheles crascens, and Anopheles dirus X (a cross between the two species) produced large numbers of P. knowlesi sporozoites. Mosquito survival to day 14, when sporozoites fill the salivary glands, averaged only 32% when donor monkeys had a parasitaemia above 2%. However, when donor monkey parasitaemia was below 2%, mosquitoes survived twice as well and contained ample sporozoites in their salivary glands. Adding methylparaben to sugar solutions did not improve survival of infected mosquitoes. Plasmodium knowlesi was very infectious, with all monkeys developing blood stage infections if one or more infected mosquitoes successfully fed. There was also a dose-response, with monkeys that received higher numbers of infected mosquito bites developing malaria sooner.ConclusionsAnopheles dirus, An. crascens and a cross between these two species all were excellent vectors for P. knowlesi. High donor monkey parasitaemia was associated with poor mosquito survival. A single infected mosquito bite is likely sufficient to infect a monkey with P. knowlesi. It is possible to efficiently challenge large groups of monkeys by mosquito bite, which will be useful for P. knowlesi vaccine studies.

The path of malaria vaccine development: challenges and perspectives

Malaria is a life-threatening disease caused by parasites of the Plasmodium genus. In many parts of the world, the parasites have developed resistance to a number of antimalarial agents. Key interventions to control malaria include prompt and effective treatment with artemisinin-based combination therapies, use of insecticidal nets by individuals at risk and active research into malaria vaccines. Protection against malaria through vaccination was demonstrated more than 30 years ago when individuals were vaccinated via repeated bites by Plasmodium falciparum-infected and irradiated but still metabolically active mosquitoes. However, vaccination with high doses of irradiated sporozoites injected into humans has long been considered impractical. Yet, following recent success using whole-organism vaccines, the approach has received renewed interest; it was recently reported that repeated injections of irradiated sporozoites increased protection in 80 vaccinated individuals. Other approaches include subunit malaria vaccines, such as the current leading candidate RTS,S (consisting of fusion between a portion of the P. falciparum-derived circumsporozoite protein and the hepatitis B surface antigen), which has been demonstrated to induce reasonably good protection. Although results have been encouraging, the level of protection is generally considered to be too low to achieve eradication of malaria. There is great interest in developing new and better formulations and stable delivery systems to improve immunogenicity. In this review, we will discuss recent strategies to develop efficient malaria vaccines.