Development of humanized mouse models to study human malaria parasite infection

Malaria is a disease caused by infection with Plasmodium parasites that are transmitted by mosquito bite. Five different species of Plasmodium infect humans with severe disease, but human malaria is primarily caused by Plasmodium falciparum. The burden of malaria on the developing world is enormous, and a fully protective vaccine is still elusive. One of the biggest challenges in the quest for the development of new antimalarial drugs and vaccines is the lack of accessible animal models to study P. falciparum infection because the parasite is restricted to the great apes and human hosts. Here, we review the current state of research in this field and provide an outlook of the development of humanized small animal models to study P. falciparum infection that will accelerate fundamental research into human parasite biology and could accelerate drug and vaccine design in the future.     

Progress towards the development of malaria vaccines

The misery and suffering caused worldwide by infection with the malaria parasite, especially Plasmodium falciparum, has been well documented. Although no licensed vaccine against malaria currently exists, progress has accelerated in recent years towards the goal of developing one. Although the complexity of the malaria parasite has made the malaria vaccine development process tenuous, advances in science and in the vaccine development process as well as increases in funding are encouraging. These advances, coupled with the results of the recent clinical trial of the vaccine candidate RTS,S, have added new vigor to the idea that a malaria vaccine is not only possible but probable.     

An update on the search for a Plasmodium vivax vaccine

Although Plasmodium falciparum is the leading cause of morbidity and mortality due to malaria worldwide, nearly 2.5 billion people, mostly outside Africa, are also at risk from malaria caused by Plasmodium vivax infection. Currently, almost all efforts to develop a malaria vaccine have focused on P. falciparum. For example, there are 23 P. falciparum vaccine candidates undergoing advanced clinical studies and only two P. vivax vaccine candidates being tested in preliminary (Phase I) clinical trials, with few others being assessed in preclinical studies. More investment and a greater effort toward the development of P. vivax vaccine components for a multi-species vaccine are required. This is mainly because of the wide geographical coexistence of both parasite species but also because of increasing drug resistance, recent observations of severe and lethal P. vivax cases and relapsing parasite behaviour. Availability of the P. vivax genome has contributed to antigen discovery but new means to test vaccines in future trials remain to be designed.     

Direct immunization of malaria DNA vaccine into the liver by gene gun protects against lethal challenge of Plasmodium berghei sporozoite

The liver is the first target organ for malaria parasites immediately after the bite of an infected mosquito. We studied local immunization of malaria DNA vaccines at the site of the liver using a gene gun as a useful tool for in vivo transfection of foreign genes. A malaria DNA vaccine consisting of the Plasmodium berghei circumsporozoite protein (PbCSP) gene plus the mouse IL-12 gene was bombarded directly by a gene gun into mouse liver once or into the skin twice. A marked protective effect was induced by gene bombardment into the liver (more than 71%) compared with that into the skin (less than 33%). A Th1-type immune response and high production of iNOS were observed in the hepatic lymphocytes from mice bombarded into the liver, resulting in more effective protection compared with those bombarded into the skin. These results provide an important implication on the development of efficient malaria vaccine strategies.     

Temperature-Dependent Pre-Bloodmeal Period and Temperature-Driven Asynchrony between Parasite Development and Mosquito Biting Rate Reduce Malaria Transmission Intensity

A mosquito needs to bite at least twice for malaria transmission to occur: once to acquire parasites and, after these parasites complete their development in their mosquito host, once to transmit the parasites to the next vertebrate host. Here we investigate the relationship between temperature, parasite development, and biting frequency in a mosquito-rodent malaria model system. We show that the pre-bloodmeal period (the time lag between mosquito emergence and first bloodmeal) increases at lower temperatures. In addition, parasite development time and feeding exhibit different thermal sensitivities such that mosquitoes might not be ready to feed at the point at which the parasite is ready to be transmitted. Exploring these effects using a simple theoretical model of human malaria shows that delays in infection and transmission can reduce the vectorial capacity of malaria mosquitoes by 20 to over 60%, depending on temperature. These delays have important implications for disease epidemiology and control, and should be considered in future transmission models.     

Malaria parasite pre-erythrocytic stage infection: gliding and hiding

In malaria, the red blood cell-infectious form of the Plasmodium parasite causes illness and the possible death of infected hosts. The initial infection in the liver caused by the mosquito-borne sporozoite parasite stage, however, causes little pathology and no symptoms. Nevertheless, pre-erythrocytic parasite stages are attracting passionate research efforts not least because they are the most promising targets for malaria vaccine development. Here, we review how the infectious sporozoite makes its way to the liver and subsequently develops within hepatocytes. We discuss the factors, both parasite and host, involved in the interactions that occur during this "silent" phase of infection.     

Antitumor effect of malaria parasite infection in a murine Lewis lung cancer model through induction of innate and adaptive immunity

Background

Lung cancer is the most common malignancy in humans and its high fatality means that no effective treatment is available. Developing new therapeutic strategies for lung cancer is urgently needed. Malaria has been reported to stimulate host immune responses, which are believed to be efficacious for combating some clinical cancers. This study is aimed to provide evidence that malaria parasite infection is therapeutic for lung cancer.

Methodology/Principal Findings

Antitumor effect of malaria infection was examined in both subcutaneously and intravenously implanted murine Lewis lung cancer (LLC) model. The results showed that malaria infection inhibited LLC growth and metastasis and prolonged the survival of tumor-bearing mice. Histological analysis of tumors from mice infected with malaria revealed that angiogenesis was inhibited, which correlated with increased terminal deoxynucleotidyl transferase-mediated (TUNEL) staining and decreased Ki-67 expression in tumors. Through natural killer (NK) cell cytotoxicity activity, cytokine assays, enzyme-linked immunospot assay, lymphocyte proliferation, and flow cytometry, we demonstrated that malaria infection provided anti-tumor effects by inducing both a potent anti-tumor innate immune response, including the secretion of IFN-γ and TNF-α and the activation of NK cells as well as adaptive anti-tumor immunity with increasing tumor-specific T-cell proliferation and cytolytic activity of CD8⁺ T cells. Notably, tumor-bearing mice infected with the parasite developed long-lasting and effective tumor-specific immunity. Consequently, we found that malaria parasite infection could enhance the immune response of lung cancer DNA vaccine pcDNA3.1-hMUC1 and the combination produced a synergistic antitumor effect.

Conclusions/Significance

Malaria infection significantly suppresses LLC growth via induction of innate and adaptive antitumor responses in a mouse model. These data suggest that the malaria parasite may provide a novel strategy or therapeutic vaccine vector for anti-lung cancer immune-based therapy.     

Malaria parasite colonisation of the mosquito midgut--placing the Plasmodium ookinete centre stage

Vector-borne diseases constitute an enormous burden on public health across the world. However, despite the importance of interactions between infectious pathogens and their respective vector for disease transmission, the biology of the pathogen in the insect is often less well understood than the forms that cause human infections. Even with the global impact of Plasmodium parasites, the causative agents of malarial disease, no vaccine exists to prevent infection and resistance to all frontline drugs is emerging. Malaria parasite migration through the mosquito host constitutes a major population bottleneck of the lifecycle and therefore represents a powerful, although as yet relatively untapped, target for therapeutic intervention. The understanding of parasite-mosquito interactions has increased in recent years with developments in genome-wide approaches, genomics and proteomics. Each development has shed significant light on the biology of the malaria parasite during the mosquito phase of the lifecycle. Less well understood, however, is the process of midgut colonisation and oocyst formation, the precursor to parasite re-infection from the next mosquito bite. Here, we review the current understanding of cellular and molecular events underlying midgut colonisation centred on the role of the motile ookinete. Further insight into the major interactions between the parasite and the mosquito will help support the broader goal to identify targets for transmission-blocking therapies against malarial disease.     

Segregation analysis detects a major gene controlling blood infection levels in human malaria.

The profound influence that the genetic makeup of the host has on resistance to malaria infection has been established in numerous animal studies. This genetic heterogeneity is one of the main causes of the difficulties in developing an effective malaria vaccine. Segregation analysis is the first step in identifying the nature of genetic factors involved in the expression of human complex diseases, as infectious diseases. To assess the role of host genes in human malaria, we performed segregation analysis of blood parasite densities in 42 Cameroonian families by using both the unified mixed model and the class D regressive model of analysis. The results provide clear evidence for the presence of a recessive major gene controlling the degree of infection in human malaria. Parameter estimates show a frequency of .44-.48 for the deleterious allele, indicating that about 21% of the population is predisposed to high levels of infection.     

DNA vaccine against malaria: a long way to go

Vaccination is the attempt to mimic certain aspects of an infection for the purpose of causing an immune response that will protect the individual from that infection. Malaria, a disease responsible for immense human suffering, is caused by infection with Plasmodium spp. parasites, which have a very complex life cycle--antigenically unique stages infect different tissues of the body. It is a parasitic disease for which no successful vaccine has been developed so far, despite considerable efforts to develop a subunit vaccine that offers protective immunity. Due to the spread of drug-resistant malaria, efforts to develop an effective vaccine have become increasingly critical. DNA vaccination provides a stable and long-lived source of protein vaccine capable of inducing both antibody- and cell-mediated immune responses to a wide variety of antigens. Injected DNA enters the cells of the host and makes the protein, which triggers the immune response. According to present needs, the flexibility of DNA vaccine technology permits the combination of multiple antigens from both the preerythrocytic and erythrocytic stages of malaria parasite. DNA vaccines with genes coding for different antigenic parts of malaria proteins have been created and presently some of these are undergoing field trials. The results from these trials will help to determine the likelihood of success of this technology in humans. This review presents an update of the studies carried out in malaria using DNA vaccine approach, the challenges, and the future prospects.     

Immunity to malaria.

Malaria remains prevalent throughout tropical and subtropical regions and almost a third of the World's population is exposed to the risk of infection. There is currently a serious resurgence of the disease in Asia and Central America. The failure of global eradication measures based upon the use of insecticides and chemotherapy has resulted from difficulties of practical implementation compounded by the spread of insecticide and drug resistance. Repeated natural infection does not produce detectable resistance to the exo-erythrocytic cycle of malaria in man. Irradiated sporzoite vaccines do, however, induce stage specific immunity in murine malaria and in a proportion of human subjects. Vaccinated individuals remain susceptible to blood stage infection which causes clinical malaria. In addition the vaccine is unstable and must be administered by intravenous inoculation. Since neither sporogonic nor exo-erythrocytic parasite development is cyclical in human malarias, there is little prospect for vaccine production through cultivation of these stages. The inhabitants of hyperendaemic areas become increasingly resistant to malaria during childhood and adolescence, through the slow development of specific, acquired immunity to asexual blood stage parasites. Immunity is mediated by antibody, which blocks merozoite invasion of red cells, as well as by cell mediated mechanisms and non-specific cytotoxic agents. Vaccination with merozoites induces long lasting immunity of broad serological specificity active against the blood-stage of the parasite. Merozoite vaccines can be preserved by freeze drying and harvested from continuous cultures of blood stage parasites. The major problem in development of a human merozoite vaccine concerns the requirement for Freund's complete adjuvant which is not acceptable for man. The effective immunity induced by vaccination contrasts with the slow development of incomplete resistance which follows repeated natural infection. The latter is associated with the generation of immune suppressor cells, lymphoid cell mitogens and soluble antigens, and in some species by the occurrence of antigenic variation--all of which may favour parasite survival. It is probable that vaccination with non-viable antigen of appropriate composition, induces immune effector processes without activating mechanisms which allow parasites to escape the consequences of immunity. Many effective vaccines such as those against measles, poliomyelitis, tetanus and rabies are commercially available but barely used in the developing world. The affected nations cannot afford their purchase, nor do the means exist for their distribution. It follows that if a safe and effective malaria vaccine were to be developed, its bulk manufacture and administration would require massive international support and cooperation.     

DNA Vaccine Against Malaria: A Long Way To Go

Vaccination is the attempt to mimic certain aspects of an infection for the purpose of causing an immune response that will protect the individual from that infection. Malaria, a disease responsible for immense human suffering, is caused by infection with Plasmodium spp. parasites, which have a very complex life cycle — antigenically unique stages infect different tissues of the body. It is a parasitic disease for which no successful vaccine has been developed so far, despite considerable efforts to develop a subunit vaccine that offers protective immunity. Due to the spread of drug-resistant malaria, efforts to develop an effective vaccine have become increasingly critical. DNA vaccination provides a stable and long-lived source of protein vaccine capable of inducing both antibody- and cell-mediated immune responses to a wide variety of antigens. Injected DNA enters the cells of the host and makes the protein, which triggers the immune response. According to present needs, the flexibility of DNA vaccine technology permits the combination of multiple antigens from both the preerythrocytic and erythrocytic stages of malaria parasite. DNA vaccines with genes coding for different antigenic parts of malaria proteins have been created and presently some of these are undergoing field trials. The results from these trials will help to determine the likelihood of success of this technology in humans. This review presents an update of the studies carried out in malaria using DNA vaccine approach, the challenges, and the future prospects.     

Malaria parasite pre-erythrocytic infection: preparation meets opportunity

For those stricken with malaria, the classic clinical symptoms are caused by the parasite's cyclic infection of red blood cells. However, this erythrocytic phase of the parasite's life cycle initiates from an asymptomatic pre-erythrocytic phase: the injection of sporozoites via the bite of a parasite-carrying Anopheline mosquito, and the ensuing infection of the liver. With the increased capabilities of studying liver stages in mice, much progress has been made elucidating the cellular and molecular basis of the parasite's progression through this bottleneck of its life cycle. Here we review relevant findings on how sporozoites prepare for infection of the liver and factors crucial to liver stage development as well as key host/parasite interactions.     

Imaging mosquito transmission of Plasmodium sporozoites into the mammalian host: Immunological implications

The malaria infection is initiated in mammals by injection of the sporozoite stage of the parasite through the bite of Plasmodium-infected, female Anopheles mosquitoes. Sporozoites are injected into extravascular portions of the skin while the mosquito is probing for a blood source. Sporozoite gliding motility allows them to locate and penetrate blood vessels of the dermis or subcutaneous tissues; once in the blood, they reach the liver, within which they continue their development. Some of the injected parasites invade dermal lymph vessels and travel to the proximal draining lymphatic node, where they interact with host immunocytes. The host responds to viable or attenuated sporozoites with antibodies directed against the immunodominant circumsporozoite protein (CSP), as well as against other sporozoite proteins. These CSP antibodies can inhibit the numbers of sporozoites injected by mosquitoes and the motility of those injected into the skin. This first phase of the immune response is followed by cell-mediated immunity involving CD8 T-cells directed against the developing liver stage of the parasite. This review discusses the early history of imaging studies, and focuses on the role that imaging has played in enabling a better understanding of both the induction and effector functions of the immune responses against sporozoites.     

Environmental constraints guide migration of malaria parasites during transmission

Migrating cells are guided in complex environments mainly by chemotaxis or structural cues presented by the surrounding tissue. During transmission of malaria, parasite motility in the skin is important for Plasmodium sporozoites to reach the blood circulation. Here we show that sporozoite migration varies in different skin environments the parasite encounters at the arbitrary sites of the mosquito bite. In order to systematically examine how sporozoite migration depends on the structure of the environment, we studied it in micro-fabricated obstacle arrays. The trajectories observed in vivo and in vitro closely resemble each other suggesting that structural constraints can be sufficient to guide Plasmodium sporozoites in complex environments. Sporozoite speed in different environments is optimized for migration and correlates with persistence length and dispersal. However, this correlation breaks down in mutant sporozoites that show adhesion impairment due to the lack of TRAP-like protein (TLP) on their surfaces. This may explain their delay in infecting the host. The flexibility of sporozoite adaption to different environments and a favorable speed for optimal dispersal ensures efficient host switching during malaria transmission.     

Molecular monitoring in malaria vaccine trials

Molecular techniques offer new approaches for malaria field trials, particularly PCR techniques, which facilitate accurate diagnosis of Plasmodium infections and increase the power of estimates of vaccine effects on malaria prevalence or incidence. Molecular methods also help to assess selective effects of vaccines. Longitudinal genotyping data can be used to initiate novel analyses of parasite dynamics, including estimates of incidence of infection with individual parasite clones and duration of infections. In addition, high-throughput methods can be used to apply these techniques routinely in randomized controlled trials, as well as programme-based evaluations of malaria control.     

Circumsporozoite protein gene from Plasmodium brasilianum. Animal reservoirs for human malaria parasites?

We describe here the sequence of the circumsporozoite protein gene of the monkey malaria parasite Plasmodium brasilianum and show that the immunodominant repeat domain is the same as that of the human malaria parasite, Plasmodium malariae. The immunodominant epitope on the surface of sporozoites of a third species of human malaria parasite has, therefore, been identified. This genetic based data and the biological similarities between P. brasilianum and P. malariae support their putative zoonotic/anthroponotic relationship. We also show that an ape malaria parasite, Plasmodium reichenowi, and the human malaria parasite, Plasmodium falciparum, have a similar relationship. The implications of these observations are discussed with respect to vaccine development.     

Plasmodium parasite proteins and the infected erythrocyte

Erythrocyte modification by malaria proteins is linked to both disease severity and infection. In this issue of Trends in Parasitology, Templeton and Deitsch, and Horrocks and Muhia discuss recent work identifying a host-targeting (HT) signal on malaria proteins. This signal predicts a secretome of 300-400 effectors for the human malaria parasite Plasmodium falciparum, vastly expanding the number of potential vaccine and drug targets. The HT signal seems to be distinct from known cellular transport signals, which suggests that it might be a novel eukaryotic secretion signal.     

Pre-erythrocytic immunity to Plasmodium falciparum: the case for an LSA-1 vaccine

A vaccine is urgently needed to stem the global resurgence of Plasmodium falciparum malaria. Vaccines targeting the erythrocytic stage are often viewed as an anti-disease strategy. By contrast, infection might be completely averted by a vaccine against the liver stage, a pre-erythrocytic stage during which the parasite multiplies 10000-fold within hepatocytes. Sterilizing immunity can be conferred by inoculating humans with irradiated pre-erythrocytic parasites, and a recombinant pre-erythrocytic vaccine partially protects humans from infection. Liver-stage antigen-1, one of a few proteins known to be expressed by liver-stage parasites, holds particular promise as a vaccine. Studies of naturally exposed populations have consistently related immune responses against this antigen to protection.