Inhibition of P. falciparum growth in human erythrocytes by monoclonal antibodies

Malaria is increasing in incidence and prevalence in most tropical areas and is a major problem for both individuals and communities. Current malaria research is aimed at developing vaccines and, for this, it may be useful to define Plasmodium antigen(s) related to the development of a protective immune response in the host. Monoclonal antibodies have recently been shown to interfere with rodent malaria infection (Plasmodium berghei) at the sporozoite or merozoite stage. We have now raised monoclonal antibodies against single antigenic determinant(s) of Plasmodium falciparum and report that some of them inhibit the growth of erythrocytic forms of P. falciparum in vitro.     

Isolate-specific S-antigen of Plasmodium falciparum contains a repeated sequence of eleven amino acids

Antibodies raised against a Plasmodium falciparum protein expressed in Escherichia coli reacted with a 220,000-molecular weight antigen of mature blood-stage parasites. The protein resembles the sporozoite surface antigen being composed of tandem repeats of 11 amino acids. However, it is isolate-specific and the encoding gene is not detectable in strains that do not express the protein.     

Cloning and expression in E. coli of the malarial sporozoite surface antigen gene from Plasmodium knowlesi

The malarial sporozoite, the infective stage found in the salivary gland of the insect vector, bears highly immunogenic surface antigen(s). Repeated exposure to irradiated sporozoites induces protection against malaria in several host species, including man. Further, monoclonal antibodies that confer passive immunity react with the immunogenic surface determinants of different sporozoite species. One approach to prevent malaria, therefore, would be to produce a vaccine that induces high titres of circulating antibodies against the sporozoite surface determinant(s). However, production of such a vaccine has not been possible since sporozoites cannot be cultivated in vitro and, therefore, only limited amounts of surface antigen may be obtained. To overcome this problem, we have prepared mRNA from Plasmodium knowlesi-infected mosquitoes to construct a cDNA library. From this library we have isolated a clone that expresses the sporozoite surface antigen as a beta-lactamase fusion protein in the plasmid pBR322. This is the first potentially protective malarial antigen to be cloned by recombinant DNA technology.     

The circumsporozoite protein is an immunodominant protective antigen in irradiated sporozoites

Malaria infection starts when mosquitoes inject sporozoites into the skin. The parasites enter the blood stream and make their way to the liver where they develop into the exo-erythrocytic forms (EEFs). Immunization with irradiated sporozoites (IrSp) leads to robust protection against malaria infection in rodents, monkeys and humans by eliciting antibodies to circumsporozoite protein (CS) that inhibit sporozoite infectivity, and T cells that destroy the EEFs. To study the role of non-CS antigens in protection, we produced CS transgenic mice that were tolerant to CS T-cell epitopes. Here we show that in the absence of T-cell-dependent immune responses to CS, protection induced by immunization with two doses of IrSp was greatly reduced. Thus, although hundreds of other Plasmodium genes are expressed in sporozoites and EEFs, CS is a dominant protective antigen. Nevertheless, sterile immunity could be obtained by immunization of CS transgenics with three doses of IrSp.     

Genetically modified Plasmodium parasites as a protective experimental malaria vaccine

Malaria is a mosquito-borne disease that is transmitted by inoculation of the Plasmodium parasite sporozoite stage. Sporozoites invade hepatocytes, transform into liver stages, and subsequent liver-stage development ultimately results in release of pathogenic merozoites. Liver stages of the parasite are a prime target for malaria vaccines because they can be completely eliminated by sterilizing immune responses, thereby preventing malarial infection. Using expression profiling, we previously identified genes that are only expressed in the pre-erythrocytic stages of the parasite. Here, we show by reverse genetics that one identified gene, UIS3 (upregulated in infective sporozoites gene 3), is essential for early liver-stage development. uis3-deficient sporozoites infect hepatocytes but are unable to establish blood-stage infections in vivo, and thus do not lead to disease. Immunization with uis3-deficient sporozoites confers complete protection against infectious sporozoite challenge in a rodent malaria model. This protection is sustained and stage specific. Our findings demonstrate that a safe and effective, genetically attenuated whole-organism malaria vaccine is possible.     

酮替酚配伍周效磺胺对红内期约氏疟原虫超微结构的影响

单独使用酮替酚或周效磺胺，红内期约氏疟原虫的超微结构受到一定程度的损伤，但配伍使用酮替酚和周效碘胺后虫体的结构变化更为迅速，给药８小时后裂殖体的形成受阻，滋养体除细胞结构逐渐消失外胞质中还出现伪食泡和逐渐增多增大的单膜或多膜空泡，给药２４小时后未查见结构完整的滋养体。     

Enhanced expression of H-2K and H-2D antigens on reticulocytes infected with Plasmodium yoelii

The 17XNL strain of Plasmodium yoelii induces a highly effective and permanent T-cell dependent immunity in mice of the CBA strain; the lethal variant P. yoelii 17XL and P. berghei (ANKA) fail to activate an effective immune response in the same host. These differences in immunogenicity are unexplained. We recently observed that in CBA/CaJ mice the intracellular blood stages of P. yoelii 17XNL were almost exclusively within reticulocytes whereas lethal P. yoelii 17XL and P. berghei (ANKA), at comparable stages of infection, were predominantly erythrocytic. Induction of a reticulocytosis converted the normally lethal P. yoelii 17XL infection into a nonlethal one, and reticulocytic P. yoelii was shown to be more immunogenic than the erythrocytic form. Since one of the differences between reticulocytes and erythrocytes that might have influenced the development of immunity was greater expression of MHC antigens of the former cell type we examined the expression of H-2K, H-2D and Ia on reticulocytes infected with P. yoelii 17XNL. These cells showed a very marked increase in H-2K and D antigen expression compared to normal reticulocytes or erythrocytes. No Ia was detected. Red blood cells (RBC) infected with lethal P. yoelii 17XL or P. berghei showed no increase in H-2K or H-2D antigen expression. Finally, the level of expression of H-2K on P. yoelii 17XNL parasitized red blood cells from different strains of mice correlated closely with the ability of these strains to control the infection.     

Malaria parasites--discovery of the early liver form

Infections of mammalian malaria parasites start when sporozoites from an infected anopheline mosquito are injected into the bloodstream of the host. The sporozoites enter the hepatocytes and become transformed into exoerythrocytic schizonts. Since the discovery of the primate parasite Plasmodium cynomolgi in monkey hepatocytes and the rodent parasite Plasmodium berghei in hamster hepatocytes, the ultrastructure of these stages has been extensively studied both in primate and rodent plasmodia. These observations relate only to the development of the exoerythrocytic schizont 25 h after sporozoite injection until the final maturation (of P. berghei) 50 h post-inoculation. Recently, we have studied the route of entry of sporozoites across the cellular lining of liver sinusoids and invasion of the liver parenchymal cells by using transmission electron microscopy. The results of these studies in combination with other physiological experiments strongly suggested that the sporozoite was initially harboured by the Kupffer cell, from which the parasite escaped into the neighbouring hepatocyte. The migration of sporozoites from liver sinusoids to hepatocytes can be achieved within a few minutes. We present here the first ultrastructural observations on the natural transformation of intrahepatocytic sporozoites into exoerythrocytic forms in vivo, using the rodent malaria parasite P. berghei in a laboratory host, the Brown Norway rat. These observations complete the search for the final link in the life cycle of malaria parasites.     

Specific role of mitochondrial electron transport in blood-stage Plasmodium falciparum

The origin of all mitochondria can be traced to the symbiotic arrangement that resulted in the emergence of eukaryotes in a world that was exclusively populated by prokaryotes. This arrangement, however, has been in continuous genetic flux: the varying degrees of gene loss and transfer from the mitochondrial genome in different eukaryotic lineages seem to signify an ongoing 'conflict' between the host and the symbiont. Eukaryotic parasites belonging to the phylum Apicomplexa provide an excellent example to support this view. These organisms contain the smallest mitochondrial genomes known, with an organization that differs among various genera; one genus, Cryptosporidium, seems to have lost the entire mitochondrial genome. Here we show that erythrocytic stages of the human malaria parasite Plasmodium falciparum seem to maintain an active mitochondrial electron transport chain to serve just one metabolic function: regeneration of ubiquinone required as the electron acceptor for dihydroorotate dehydrogenase, an essential enzyme for pyrimidine biosynthesis. Transgenic P. falciparum parasites expressing Saccharomyces cerevisiae dihydroorotate dehydrogenase, which does not require ubiquinone as an electron acceptor, were completely resistant to inhibitors of mitochondrial electron transport. Maintenance of mitochondrial membrane potential, however, was essential in these parasites, as indicated by their hypersensitivity to proguanil, a drug that collapsed the membrane potential in the presence of electron transport inhibitors. Thus, acquisition of just one enzyme can render mitochondrial electron transport nonessential in erythrocytic stages of P. falciparum.     

疟疾疫苗的研究策略

从疟原虫的不同发育时期、不同的疫苗成份和宿主的遗传基因限制性等方面，深入研究抗疟疾疫苗。作用于红细胞前期的疟疾疫苗主要是抑制疟疾的临床发作，控制疟疾的传播；作用于红细胞期的疟疾疫苗诱导宿主体液免疫系统，产生特异性抗体，抑制疟原虫侵入和感染红细胞，达到减少疟原虫虫荷，降低疟疾的发病率和死亡率。作用于疟原虫有性生殖时期，控制疟疾传播的疟疾疫苗，其在于控制一个地区疟原虫的感染率和疟疾发病率，但对已感染疟原虫个体的免疫保护作用意义不大。在设计疟疾疫苗的过程中，必须克服不同个体的遗传基因限制性问题。由于疟原虫生活史的复杂性，同时也必须考虑到疟原虫不同发育阶段抗原成份的复杂性。     

A rhoptry-protein-associated mechanism of clonal phenotypic variation in rodent malaria

The recognition and invasion of host cells are mediated by components of the apical complex of the ookinete, sporozoite and merozoite stages of Plasmodium parasites. The paired rhoptries (organelles involved in host-cell recognition) in the apical complex contain many proteins of as-yet unknown function. In the rodent malaria agent P. yoelii yoelii, a multigene family codes for merozoite rhoptry proteins of relative molecular mass 235,000 (p235 proteins); these proteins are thought to determine the subset of erythrocytes that the parasites invade. Further support for this idea came from the identification of a region in p235 with weak but significant homology to reticulocyte-binding protein-2 of P. vivax and the demonstration that at least one p235 member binds to the erythrocyte surface membrane. Here, using single, micromanipulated P.y.yoelii parasites, we describe a new mechanism of gene expression by which the merozoites originating from a single schizont each express a distinct member of this multigene family. We propose that this new type of clonal phenotypic variation provides the parasite with a survival strategy in the mammalian host; this strategy contributes to the observed chronicity of malarial infections. This phenomenon is genetically and functionally distinct from classical antigenic variation, which is mediated by the var multigene family of P. falciparum.     

Vaccination with purified microgamete antigens prevents transmission of rodent malaria

Malaria vaccination with preparations of microgametes has been shown to inhibit transmission of Plasmodium spp. to the mosquito vectors of avian, rodent and simian parasites. This transmission-blocking immunity results from the induction of microgamete-agglutinating antibodies in the vaccinated host which, when ingested in a mosquito blood meal, react with exflagellating microgametes in the midgut to prevent fertilization and oocyst development. Here we have passively transferred the immunity with gamete-specific monoclonal antibodies raised against the rodent malaria parasite Plasmodium yoelii nigeriensis, and an IgG2a isotype monoclonal antibody from a hybridoma cell line, PYG-1, has been used to identify the target antigens on the gametes and to affinity-purify sufficient quantities of specific gamete antigen to facilitate vaccination studies. This transmission-blocking monoclonal antibody immunoprecipitated microgamete antigens of apparent relative molecular mass (Mr), 67K (67,000), 59K, 57K, 42K and 35K. Immunization of mice with these proteins before infection and mosquito feeding led to a 85-99.7% reduction in transmission to the mosquito vector; vaccination via intravenous or intramuscular routes was equally effective and did not require an adjuvant.     

Direct access to serum macromolecules by intraerythrocytic malaria parasites.

Trafficking pathways in malaria-infected erythrocytes are complex because the internal parasite is separated from the serum by the erythrocyte and parasitophorous vacuolar membranes. Intraerythrocytic Plasmodium falciparum parasites can endocytose dextrans, protein A and an IgG2a antibody. Here we show that these macromolecules do not cross the erythrocyte or parasitophorous vacuolar membranes, but rather gain direct access to the aqueous space surrounding the parasite through a parasitophorous duct. Evidence for this structure includes visualization of membranes that are continuous between the parasitophorous vacuolar and erythrocyte membranes, and surface labelling of the parasite with fluorescent macromolecules under conditions that block endocytosis. The parasite can internalize by fluid-phase endocytosis macromolecules from the aqueous compartment surrounding it. Thus, surface antigens on trophozoites and schizonts should be considered as targets for antibody-directed parasiticidal agents.     

T cells can present antigens such as HIV gp120 targeted to their own surface molecules

To trigger class II-restricted T cells, antigen presenting cells have to capture antigens, process them and display their fragments in association with class II molecules. In most species, activated T cells express class II molecules; however, no evidence has been found that these cells can present soluble antigens. This failure may be due to the inefficient capture, processing or display of antigens in a stimulatory form by T-cells. The capture of a soluble antigen, which is achieved by nonspecific mechanisms in macrophages and dendritic cells, can be up to 10(3) times more efficient in the presence of surface receptors, such as surface immunoglobulin on B cells that specifically bind antigen with high affinity. We asked whether T cells would be able to present soluble antigens that bind to their own surface molecules. Here we show that such antigens can be effectively processed and presented by both CD4+- and CD8+-bearing human T cells. This indicates that T cells are fully capable of processing and displaying antigens and are mainly limited in antigen presentation by their inefficiency at antigen capture.     

Gametocytogenesis by malaria parasites in continuous culture

Asexual proliferation of malaria parasites proceeds by multiplication of the parasites within red cells. Following rupture of the host cells the released merozoites re-invade other red cells. On re-invasion, a proportion of merozoites become, not asexual parasites but gametocytes, the sexual stages infective to the mosquito vectors. Conversion of asexual parasites to gametocytes occurs not only during natural infections but also in continuous in vitro culture as reported first by Trager and Jensen and by others. We showed previously that the proportion of early intra-erythrocytic stages (ring stages) of Plasmodium falciparum which developed into gametocytes in culture was influenced by culture conditions. Gametocyte formation was rare in conditions supporting rapid proliferation but frequent when parasite densisites were static. We now show that nearly 100% of ring stages develop into gametocytes in response to 1mM cyclic AMP in static cultures whereas in rapidly growing cultures few rings become gametocytes in response to cyclic AMP.     

Immune sera recognize on erythrocytes Plasmodium falciparum antigen composed of repeated amino acid sequences

Protective immune responses against the asexual stages of the human malaria parasite, Plasmodium falciparum, are most probably directed against exposed antigenic determinants on the surface of the free merozoite or the infected red blood cell, and therefore antigens in these locations are candidates for testing as components of a defined molecular vaccine. To facilitate the search for such antigens, we recently developed a method for the expression of P. falciparum proteins in Escherichia coli as fused polypeptides. Many clones producing antigens were detected by screening with immune human sera. We show here that antibodies against the fused polypeptide expressed by one such clone react with a P. falciparum protein that is synthesized late in schizogony and is later present on the surface of the ring-infected erythrocyte. The protein is composed of repeating subunits of 8, 4 and 3 amino acids and is present in all isolates of P. falciparum examined.     

Immunization of Aotus monkeys with recombinant proteins of an erythrocyte surface antigen of Plasmodium falciparum

Recent studies have identified and characterized a ring-infected erythrocyte surface antigen (RESA) of the human malaria parasite Plasmodium falciparum with a relative molecular mass (Mr) of approximately 155,000 (refs 1-7). RESA is localized in the micronemes of merozoites and also the membrane of red cells infected with ring-stage parasites. It is thought to be released through the apical pore from the rhoptry at the time of merozoite invasion. Because antibodies directed against this antigen strongly inhibit parasite growth in vitro, RESA may be useful in developing a vaccine against this parasite Here we describe an immunization trial using Aotus monkeys and Escherichia coli-derived fused polypeptides corresponding to various regions of the RESA molecule. Some monkeys in all test groups, but not in the control group, were protected against overwhelming infection. Strikingly, protection correlated with antibody responses to either of two different repetitive sequences in RESA.     

Transient cross-reactive immune responses can orchestrate antigenic variation in malaria

The malaria parasite Plasmodium falciparum has evolved to prolong its duration of infection by antigenic variation of a major immune target on the surface of the infected red blood cell. This immune evasion strategy depends on the sequential, rather than simultaneous, appearance of immunologically distinct variants. Although the molecular mechanisms by which a single organism switches between variants are known in part, it remains unclear how an entire population of parasites within the host can synchronize expression to avoid rapidly exhausting the variant repertoire. Here we show that short-lived, partially cross-reactive immune responses to parasite-infected erythrocyte surface antigens can produce a cascade of sequentially dominant antigenic variants, each of which is the most immunologically distinct from its preceding types. This model reconciles several previously unexplained and apparently conflicting epidemiological observations by demonstrating that individuals with stronger cross-reactive immune responses can, paradoxically, be more likely to sustain chronic infections. Antigenic variation has always been seen as an adaptation of the parasite to evade host defence: we show that the coordination necessary for the success of this strategy might be provided by the host.