Arrest in the Liver — A Genetically Defined Malaria Vaccine?
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《新英格兰医药杂志》
Malaria is a disease with far-reaching effects: 40 percent of the world's population is at risk for malarial infection, approximately 300 million to 400 million cases occur annually, and each year, 1 million to 2 million people — predominantly young children — die of malaria. And yet there are no vaccines for parasitic diseases that affect humans, let alone a vaccine for malaria.
Sporozoites, the infectious stage of the malaria parasite plasmodium, are small, slender, unicellular organisms that mature in the salivary glands of anopheline mosquitoes. Exposure to radiation-attenuated sporozoites, however, can elicit a cell- and antibody-mediated immune response sufficient to prevent malarial infection in humans (referred to as sterile immunity — i.e., total absence of the blood-stage parasites responsible for clinical disease). Volunteers immunized with radiation-attenuated sporozoites of Plasmodium falciparum and P. vivax, delivered by the bites of mosquitoes, were protected against infection when challenged with viable sporozoites.1,2 But there are logistical and technical obstacles to using sporozoites as vaccines, such as the need to attenuate infectivity by means of irradiation. Mueller and colleagues recently showed that genetically modified sporozoites induce complete immunity in a mouse model of malaria, thus overcoming these obstacles.3
Having previously identified plasmodium antigens unique to the parasites' preerythrocytic stage, the authors engineered P. berghei parasites lacking the uis3 (up-regulated in infective sporozoites) gene.4 All mice immunized with multiple doses of uis3– sporozoites were fully protected against malaria when challenged with an intravenous injection of wild-type sporozoites or by the bites of infected mosquitoes. Just as important was the finding that the uis3– sporozoites elicited immunity when injected subcutaneously — a route acceptable for the delivery of vaccines in humans. Moreover, attenuated, whole-organism vaccines do not require exogenous adjuvants. The limited repertoire of adjuvants acceptable for human use impedes the development of "subunit" vaccines, in which single proteins or domains of proteins are the vaccinating agent.
The ability to elicit sterile immunity to a eukaryotic parasite such as plasmodium is remarkable, given the complex life cycle of the parasite and the unique journey that sporozoites must take before reaching the liver, their initial site of replication in the host. While probing the skin in search of a blood meal, plasmodium-infected mosquitoes introduce saliva containing a small number of sporozoites into the subcutaneous tissue. The parasites leave the bite site, penetrate a capillary, and travel through the bloodstream to the liver.5 After entering a liver sinusoid, sporozoites are thought to recognize extracellular-matrix proteoglycans that protrude into the sinusoidal lumen and bind sporozoite surface proteins, including one called the circumsporozoite protein. The parasites then glide along the sinusoidal cell layer until they encounter a Kupffer's cell (a type of macrophage specific to the liver), which they invade, and thus gain access to hepatocytes (Figure 1). Once inside the liver tissue, sporozoites pass through several hepatocytes, fatally wounding them in the process before finally settling down in a single hepatocyte to mature and then differentiate into thousands of merozoites, which are capable of infecting red cells.
Figure 1. Life Cycle of Plasmodium in the Liver and Blood.
Malaria sporozoites enter a liver lobule through either the portal vein or the hepatic artery and glide along the sinusoidal cell layer by binding to extracellular-matrix proteoglycans secreted by stellate cells. Liver sinusoids are lined by endothelial cells containing fenestrations that allow substances destined for removal and detoxification by the liver access to hepatocytes. Interspersed in the layer of sinusoidal endothelia are Kupffer's cells, resident macrophages of the liver, which are responsible for the clearance of foreign particles from the blood. Sporozoites use Kupffer's cells to gain access to hepatocytes (Panel A). The sporozoites then migrate through several hepatocytes before settling into one and undergoing further development (Panels B and C). After gradually growing larger than their host cell during the liver stage of infection (Panels D and E), the parasites differentiate into thousands of merozoites (Panel F), which are released into the circulating blood (Panel G), where they rapidly infect red cells (Panel H). Maturation of the blood-stage schizonts, resulting in the periodic amplification and release of merozoites from red cells, leads to clinical malaria. Using a mouse model, Mueller et al.3 recently showed that the uis3 gene, which is expressed by sporozoites and early liver stages, is critical to the development and maturation of the parasite in the liver. Unable to differentiate into merozoites (red bar between Panels B and C), uis3– parasites act as a vaccine and elicit immunity. Similarly, radiation-attenuated sporozoites also fail to produce merozoites and generate sterile immunity.
Radiation-attenuated sporozoites and uis3– sporozoites are able to infect the liver but cannot undergo nuclear division. Consequently, maturation and differentiation into the merozoite stage are blocked, and the infection fails to proceed to clinical malaria. Studies of hosts immunized with radiation-attenuated sporozoites have shown that antibodies against a circumsporozoite protein are elicited. After the host has been exposed to the bite of an infected mosquito, these antibodies inhibit the motility of sporozoites, immobilizing the parasites in the skin, blocking liver infection, and enhancing opsonization and phagocytosis of the sporozoites. If sporozoites evade such antibodies, however, they can invade hepatocytes, where humoral immunity is no longer effective, but the circumsporozoite protein and other newly expressed liver-stage antigens provide targets for protective CD4+ and CD8+ T-cell–mediated immunity.
Mueller et al. exploited the P. berghei genome to construct a genetically modified parasite that undergoes normal sporozoite development but abortive hepatic-stage development.3 In so doing, they provide a means of delivering preerythrocytic parasite antigens in their native configuration. Although this particular example of reverse genetics has advanced hopes that an attenuated-sporozoite vaccine may soon be developed, important challenges remain. Critical questions regarding the longevity and mechanism of immunity induced by genetically attenuated uis3– parasites must be answered. Other challenges include the preparation of sterile parasites free of mosquito debris (currently, sporozoites are obtained by dissecting the salivary glands of infected mosquitoes), the development of efficient cryopreservation protocols, and the route of immunization. In the meantime, study of the uis3– parasites may help to identify antigens expressed in the early stages of liver infection that are critical to the development of cell-mediated immunity — thereby speeding the design of subunit vaccines, which are more amenable to mass production.
Source Information
From the Department of Medical and Molecular Parasitology, New York University School of Medicine, New York.
References
Clyde DF, Most H, McCarthy VC, Vanderberg JP. Immunization of man against sporozoite-induced falciparum malaria. Am J Med Sci 1973;266:169-177.
Luke TC, Hoffman SL. Rationale and plans for developing a non-replicating, metabolically active, radiation-attenuated Plasmodium falciparum sporozoite vaccine. J Exp Biol 2003;206:3803-3808.
Mueller AK, Labaied M, Kappe SH, Matuschewski K. Genetically modified Plasmodium parasites as a protective experimental malaria vaccine. Nature 2005;433:164-167.
Matuschewski K, Ross J, Brown SM, Kaiser K, Nussenzweig V, Kappe SH. Infectivity-associated changes in the transcriptional repertoire of the malaria parasite sporozoite stage. J Biol Chem 2002;277:41948-41953.
Frevert U. Sneaking in through the back entrance: the biology of malaria liver stages. Trends Parasitol 2004;20:417-424.(Ute Frevert, D.V.M., Ph.D)
Sporozoites, the infectious stage of the malaria parasite plasmodium, are small, slender, unicellular organisms that mature in the salivary glands of anopheline mosquitoes. Exposure to radiation-attenuated sporozoites, however, can elicit a cell- and antibody-mediated immune response sufficient to prevent malarial infection in humans (referred to as sterile immunity — i.e., total absence of the blood-stage parasites responsible for clinical disease). Volunteers immunized with radiation-attenuated sporozoites of Plasmodium falciparum and P. vivax, delivered by the bites of mosquitoes, were protected against infection when challenged with viable sporozoites.1,2 But there are logistical and technical obstacles to using sporozoites as vaccines, such as the need to attenuate infectivity by means of irradiation. Mueller and colleagues recently showed that genetically modified sporozoites induce complete immunity in a mouse model of malaria, thus overcoming these obstacles.3
Having previously identified plasmodium antigens unique to the parasites' preerythrocytic stage, the authors engineered P. berghei parasites lacking the uis3 (up-regulated in infective sporozoites) gene.4 All mice immunized with multiple doses of uis3– sporozoites were fully protected against malaria when challenged with an intravenous injection of wild-type sporozoites or by the bites of infected mosquitoes. Just as important was the finding that the uis3– sporozoites elicited immunity when injected subcutaneously — a route acceptable for the delivery of vaccines in humans. Moreover, attenuated, whole-organism vaccines do not require exogenous adjuvants. The limited repertoire of adjuvants acceptable for human use impedes the development of "subunit" vaccines, in which single proteins or domains of proteins are the vaccinating agent.
The ability to elicit sterile immunity to a eukaryotic parasite such as plasmodium is remarkable, given the complex life cycle of the parasite and the unique journey that sporozoites must take before reaching the liver, their initial site of replication in the host. While probing the skin in search of a blood meal, plasmodium-infected mosquitoes introduce saliva containing a small number of sporozoites into the subcutaneous tissue. The parasites leave the bite site, penetrate a capillary, and travel through the bloodstream to the liver.5 After entering a liver sinusoid, sporozoites are thought to recognize extracellular-matrix proteoglycans that protrude into the sinusoidal lumen and bind sporozoite surface proteins, including one called the circumsporozoite protein. The parasites then glide along the sinusoidal cell layer until they encounter a Kupffer's cell (a type of macrophage specific to the liver), which they invade, and thus gain access to hepatocytes (Figure 1). Once inside the liver tissue, sporozoites pass through several hepatocytes, fatally wounding them in the process before finally settling down in a single hepatocyte to mature and then differentiate into thousands of merozoites, which are capable of infecting red cells.
Figure 1. Life Cycle of Plasmodium in the Liver and Blood.
Malaria sporozoites enter a liver lobule through either the portal vein or the hepatic artery and glide along the sinusoidal cell layer by binding to extracellular-matrix proteoglycans secreted by stellate cells. Liver sinusoids are lined by endothelial cells containing fenestrations that allow substances destined for removal and detoxification by the liver access to hepatocytes. Interspersed in the layer of sinusoidal endothelia are Kupffer's cells, resident macrophages of the liver, which are responsible for the clearance of foreign particles from the blood. Sporozoites use Kupffer's cells to gain access to hepatocytes (Panel A). The sporozoites then migrate through several hepatocytes before settling into one and undergoing further development (Panels B and C). After gradually growing larger than their host cell during the liver stage of infection (Panels D and E), the parasites differentiate into thousands of merozoites (Panel F), which are released into the circulating blood (Panel G), where they rapidly infect red cells (Panel H). Maturation of the blood-stage schizonts, resulting in the periodic amplification and release of merozoites from red cells, leads to clinical malaria. Using a mouse model, Mueller et al.3 recently showed that the uis3 gene, which is expressed by sporozoites and early liver stages, is critical to the development and maturation of the parasite in the liver. Unable to differentiate into merozoites (red bar between Panels B and C), uis3– parasites act as a vaccine and elicit immunity. Similarly, radiation-attenuated sporozoites also fail to produce merozoites and generate sterile immunity.
Radiation-attenuated sporozoites and uis3– sporozoites are able to infect the liver but cannot undergo nuclear division. Consequently, maturation and differentiation into the merozoite stage are blocked, and the infection fails to proceed to clinical malaria. Studies of hosts immunized with radiation-attenuated sporozoites have shown that antibodies against a circumsporozoite protein are elicited. After the host has been exposed to the bite of an infected mosquito, these antibodies inhibit the motility of sporozoites, immobilizing the parasites in the skin, blocking liver infection, and enhancing opsonization and phagocytosis of the sporozoites. If sporozoites evade such antibodies, however, they can invade hepatocytes, where humoral immunity is no longer effective, but the circumsporozoite protein and other newly expressed liver-stage antigens provide targets for protective CD4+ and CD8+ T-cell–mediated immunity.
Mueller et al. exploited the P. berghei genome to construct a genetically modified parasite that undergoes normal sporozoite development but abortive hepatic-stage development.3 In so doing, they provide a means of delivering preerythrocytic parasite antigens in their native configuration. Although this particular example of reverse genetics has advanced hopes that an attenuated-sporozoite vaccine may soon be developed, important challenges remain. Critical questions regarding the longevity and mechanism of immunity induced by genetically attenuated uis3– parasites must be answered. Other challenges include the preparation of sterile parasites free of mosquito debris (currently, sporozoites are obtained by dissecting the salivary glands of infected mosquitoes), the development of efficient cryopreservation protocols, and the route of immunization. In the meantime, study of the uis3– parasites may help to identify antigens expressed in the early stages of liver infection that are critical to the development of cell-mediated immunity — thereby speeding the design of subunit vaccines, which are more amenable to mass production.
Source Information
From the Department of Medical and Molecular Parasitology, New York University School of Medicine, New York.
References
Clyde DF, Most H, McCarthy VC, Vanderberg JP. Immunization of man against sporozoite-induced falciparum malaria. Am J Med Sci 1973;266:169-177.
Luke TC, Hoffman SL. Rationale and plans for developing a non-replicating, metabolically active, radiation-attenuated Plasmodium falciparum sporozoite vaccine. J Exp Biol 2003;206:3803-3808.
Mueller AK, Labaied M, Kappe SH, Matuschewski K. Genetically modified Plasmodium parasites as a protective experimental malaria vaccine. Nature 2005;433:164-167.
Matuschewski K, Ross J, Brown SM, Kaiser K, Nussenzweig V, Kappe SH. Infectivity-associated changes in the transcriptional repertoire of the malaria parasite sporozoite stage. J Biol Chem 2002;277:41948-41953.
Frevert U. Sneaking in through the back entrance: the biology of malaria liver stages. Trends Parasitol 2004;20:417-424.(Ute Frevert, D.V.M., Ph.D)