Plasmodium falciparum: release of circumsporozoite protein by sporozoites in the mosquito vector.

The release of circumsporozoite (CS) protein by Plasmodium falciparum sporozoites was investigated to identify factors regulating this process within infected Anopheles gambiae mosquitoes. The potential for sporozoites to release CS protein in vitro was not dependent upon their site-specific developmental stage (i.e., mature oocysts, hemolymph, salivary glands), their duration in the vector, or their exposure to mosquito-derived components such as salivary glands or hemolymph. The capacity of sporozoites to release CS protein was depressed by mosquito blood feeding during periods of sporozoite migration to the salivary glands, but the effect was only temporary and those sporozoites already in the glands were not affected. Free CS protein in the salivary glands was present in 93.3% of 45 infective mosquitoes. Sporozoites from these same, individual mosquitoes were also tested in vitro for CS protein release. In both cases, the amount of soluble CS protein increased as a function of sporozoite density but the total amount of CS protein per sporozoite became progressively less with increasing numbers of sporozoites. Further experiments showed that sporozoite contact with increasing amounts of soluble CS protein caused a down-regulation of CS protein release. Thus, a primary factor regulating the production and release of CS protein by sporozoites is their contact with soluble CS protein within the mosquito.     

Binding and invasion of liver cells by Plasmodium falciparum sporozoites. Essential involvement of the amino terminus of circumsporozoite protein.

Plasmodium sporozoites display circumsporozoite (CS) protein on their surface, which is involved in the attachment of sporozoites to liver cells. CS protein is a member of the thrombospondin type I repeat (TSR) domain family and possess a single copy of TSR domain toward its carboxyl terminus. We show by a direct measurement the correlation between the binding activity of various segments of the CS protein and their ability to inhibit the invasion of liver cells by the sporozoites. We made eight truncated versions of Plasmodium falciparum CS protein to elucidate the role of various regions in the binding and invasion process. Deletion of the TSR domain actually enhanced binding activity by 2-3-fold without the loss of receptor specificity, indicating that TSR may not be the only domain in defining the specificity of binding. These same deletions blocked invasion of live sporozoites more efficiently than proteins that include the TSR domain. Deletion of as little as six amino acids from amino terminus of the protein, however, renders it incapable of binding to liver cells and as an inhibitor of sporozoite invasion. Hence, the binding of CS protein to liver cells and its ability to inhibit the invasion process are affected in a parallel manner, both positively and negatively, by sequence changes in the encoded CS gene. This indicates that both assays are measuring interrelated phenomenon and points to the essential involvement for the amino-terminal portion of the CS protein in these processes.     

Motility and infectivity of Plasmodium berghei sporozoites expressing avian Plasmodium gallinaceum circumsporozoite protein

Avian and rodent malaria sporozoites selectively invade different vertebrate cell types, namely macrophages and hepatocytes, and develop in distantly related vector species. To investigate the role of the circumsporozoite (CS) protein in determining parasite survival in different vector species and vertebrate host cell types, we replaced the endogenous CS protein gene of the rodent malaria parasite Plasmodium berghei with that of the avian parasite P. gallinaceum and control rodent parasite P. yoelii. In anopheline mosquitoes, P. berghei parasites carrying P. gallinaceum and rodent parasite P. yoelii CS protein gene developed into oocysts and sporozoites. Plasmodium gallinaceum CS expressing transgenic sporozoites, although motile, failed to invade mosquito salivary glands and to infect mice, which suggests that motility alone is not sufficient for invasion. Notably, a percentage of infected Anopheles stephensi mosquitoes showed melanotic encapsulation of late stage oocysts. This was not observed in control infections or in A. gambiae infections. These findings shed new light on the role of the CS protein in the interaction of the parasite with both the mosquito vector and the rodent host.     

Plasmodium falciparum: glycosylation status of Plasmodium falciparum circumsporozoite protein expressed in the baculovirus system

We expressed the main surface antigen of Plasmodium falciparum sporozoites, the circumsporozoite protein (CSP), in High Five (Trichoplusia ni) insect cells using the baculovirus system. Significant amounts of the recombinant protein could be obtained, as judged by SDS-PAGE, Western blot, and immunofluorescence analysis. The cellular localization for recombinant CSP was determined by immunofluorescence. The high fluorescence signal of the permeabilized cells, relative to that of fixed nonpermeabilized cells, revealed a clear intracellular localization of this surface antigen. Analysis of possible posttranslational modifications of CSP showed that this recombinant protein is only N-glycosylated in the baculovirus system. Although DNA-sequence analysis revealed a GPI-cleavage/attachment site, no GPI anchor could be demonstrated. These analyses show that the glycosylation status of this recombinant protein may not reflect its native form in P. falciparum. The impact of these findings on vaccine development will be discussed.     

Exit of Plasmodium sporozoites from oocysts is an active process that involves the circumsporozoite protein

Plasmodium sporozoites develop within oocysts residing in the mosquito midgut. Mature sporozoites exit the oocysts, enter the hemolymph, and invade the salivary glands. The circumsporozoite (CS) protein is the major surface protein of salivary gland and oocyst sporozoites. It is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. CS protein contains a conserved motif of positively charged amino acids: region II-plus, which has been implicated in the initial stages of sporozoite invasion of hepatocytes. We investigated the function of region II-plus by generating mutant parasites in which the region had been substituted with alanines. Mutant parasites produced normal numbers of sporozoites in the oocysts, but the sporozoites were unable to exit the oocysts. In in vitro as well, there was a profound delay, upon trypsin treatment, in the release of mutant sporozoites from oocysts. We conclude that the exit of sporozoites from oocysts is an active process that involves the region II-plus of CS protein. In addition, the mutant sporozoites were not infective to young rats. These findings provide a new target for developing reagents that interfere with the transmission of malaria.     

Plasmodium malariae: distribution of circumsporozoite protein in midgut oocysts and salivary gland sporozoites

The distribution of the circumsporozoite protein within developing Plasmodium malariae oocysts and salivary gland sporozoites was examined by immunoelectron microscopy using protein A-gold and a monoclonal antibody specific for the CS protein of P. malariae. Gold particles were found along the capsule of immature oocysts but rarely within the cytoplasm. Gold label was detected on the inner surface of peripheral vacuoles during oocyst maturation and the plasma membrane of the sporoblast. Salivary gland sporozoites and budding sporozoites in mature oocysts were labeled uniformly on the outer surface of their plasma membranes. The surface of sporozoites that ruptured into midgut epithelial cells were entirely covered with gold particles. No label was seen on the surface of sporozoites which ruptured into the midgut lumen. In addition, a rabbit polyclonal antibody against repeat a region of P. brasilianum CS protein reacted with P. malariae sporozoites.     

Human T cells recognize polymorphic and non-polymorphic regions of the Plasmodium falciparum circumsporozoite protein.

In order to characterize T cell epitopes in the Plasmodium falciparum circumsporozoite (CS) protein sequence, we isolated T cell clones, from non-immune donors, which reacted with synthetic peptides corresponding to two predicted CS protein T cell epitopes. Peptide CS.T3 (corresponding to a non-polymorphic region of the CS protein, residues 378-398) was recognized in association with either DR2 or DRw9 restriction elements. T cell clones recognizing CS.T3 also reacted with the sporozoite-derived CS protein. Peptide CS.T2 corresponds to a polymorphic region (residues 325-341) of the CS protein. Unlike the CS.T3-specific clones, the CS.T2-specific clones did not recognize the CS protein. Since the CS.T2 peptide includes residues which are polymorphic in different P. falciparum isolates, we investigated whether these residues were critical for recognition of the peptide. We show here that a single amino acid substitution at a position of the CS protein which shows genetic polymorphism affects recognition of the sequence by human T cells. The implications of these data for malaria vaccine development are discussed.     

Ultrastructural localization of Plasmodium falciparum circumsporozoite protein in newly invaded hepatoma cells

The fate and disposition of the circumsporozoite (CS) protein of Plasmodium falciparum was investigated during hepatoma cell invasion with several sera raised against defined CS peptides, including both repeat and nonrepeat regions spanning approximately 60% of the P. falciparum CS gene product. Distribution of the protein, as revealed by immunoelectron microscopy, was limited to the surface of the sporozoite both before and after invasion. In particular, no CS protein antigen was detected in association with either the parasitophorous vacuole membrane or the host cell surface.     

Secretion of Plasmodium falciparum rhoptry protein into the plasma membrane of host erythrocytes

The rhoptry is an organelle of the malarial merozoite which has been suggested to play a role in parasite invasion of its host cell, the erythrocyte. A monoclonal antibody selected for reactivity with this organelle identifies a parasite synthesized protein of 110 kD. From biosynthetic labeling experiments it was demonstrated that the protein is synthesized midway through the erythrocytic cycle (the trophozoite stage) but immunofluorescence indicates the protein is not localized in the organelle until the final stage (segmenter stage) of intraerythrocytic development. Immunoelectron microscopy shows that the protein is localized in the matrix of the rhoptry organelle and on membranous whorls secreted from the merozoite. mAb recognition of the protein is dithiothreitol (DTT) labile, indicating that the conformation of the epitope is dependent on a disulfide linkage. During erythrocyte reinvasion by the extracellular merozoite, immunofluorescence shows the rhoptry protein discharging from the merozoite and spreading around the surface of the erythrocyte. The protein is located in the plasma membrane of the newly invaded erythrocyte. These studies suggest that the 110-kD rhoptry protein is inserted into the membrane of the host erythrocyte during merozoite invasion.     

An asparagine-rich protein from blood stages of Plasmodium falciparum shares determinants with sporozoites.

We describe a cDNA clone derived from mRNA of asexual blood-stages of the malaria parasite Plasmodium falciparum. This clone, designated Ag319, expresses a P.falciparum antigen fused to beta-galactosidase in Escherichia coli. Human antibodies from Papua New Guinea were affinity-purified by adsorption to extracts of Ag319 immobilized on CNBr-Sepharose. The antibodies reacted predominantly with P. falciparum polypeptides of Mr 220,000 and 160,000, and a number of ill-defined lower molecular weight species. Antibodies reacted in indirect immunofluorescence with all asexual blood-stages although the antigen appeared to be most abundance in the schizont. Surprizingly the antibodies also reacted with sporozoites. The amino acid sequence predicted from the complete nucleotide sequence of this clone is remarkable because 40% of the residues are Asn, and so the antigen has been termed the Asparagine-Rich Protein (ARP). Like other P. falciparum antigens, ARP contains tandemly repetitive sequences, based on the tetrapeptide Asn-Asn-Asn-Met and we have confirmed that these represent natural epitopes by reaction of the corresponding synthetic peptides with human antibodies. Surprisingly, ARP is also rich in Asn outside the tandem repeats.     

Plasmodium gallinaceum: antibodies to circumsporozoite protein prevent sporozoites from invading the salivary glands of Aedes aegypti.

A circumsporozoite protein-specific monoclonal antibody (N2H6D5) was injected into malaria-infected mosquitoes to determine its effect on the sporogonic cycle. After injection of antibody into mosquitoes (100 ng each), positive immunofluorescence (measured on air-dried sporozoites) reactions in hemolymph extracts were observed at a dilution of 1:1000. At 72 hr postinjection the levels dropped to 1:10. Sporozoites coinjected with antibody did not invade the salivary glands. In naturally infected mosquitoes, sporozoites were released over a period of 3 to 4 days. Therefore, mosquitoes were injected twice. The first injection was a day before the beginning of sporozoite release and the second, 2 days later. Sporozoite invasion of the salivary glands was assessed 3 days after the second injection, by microscopic examination of dissected glands. At this stage, all oocysts had completed maturation and released the sporozoites. Salivary gland infections were totally prevented in mosquitoes given two injections of 100 ng N2H6D5. Hence, sustained presence of anti-circumsporozoite antibodies in the hemolymph can render female Aedes aegypti refractory to Plasmodium gallinaceum.     

Naturally acquired CD8+ cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein.

In rodent malaria model systems, protective immunity induced by immunization with irradiated sporozoites is eliminated by in vivo depletion of CD8+ T cells, and adoptive transfer of CTL clones against the circumsporozoite protein protects against malaria. We recently demonstrated that volunteers immunized with irradiated Plasmodium falciparum sporozoites produce CTL against peptide 368-390 of the P. falciparum circumsporozoite protein. To determine whether natural exposure to malaria induced similar CTL, we studied 11 adult, male, life-long residents of a highly malarious area of Kenya, who were selected because their lymphocytes had been shown to proliferate after stimulation with peptides 361-380, 371-390, or 368-390 and because nine had been resistant to malaria in previous studies. In four of the 11 individuals there was peptide-specific, genetically restricted, CTL activity. In all four individuals, this activity was unaffected by depletion of CD4+ T cells. In three volunteers the activity was eliminated or reduced by depletion of CD8+ T cells; in the fourth volunteer the CD8+ T cell depletion was uninterpretable. This first demonstration of CD8+ T cell, genetically restricted, Ag-specific CTL against a malaria protein among individuals exposed to endemic malaria provides a foundation for studying the relationship between circulating CTL and resistance to malaria infection.     

Plasmodium falciparum: immunogenicity of circumsporozoite protein constructs produced in Escherichia coli

The immunogenicity of Plasmodium falciparum recombinant circumsporozoite protein constructs R16tet32, R32tet32, and R48tet32 in mice was examined by measuring antibody responses by enzyme linked immunosorbent assay, immunofluorescence, circumsporozoite precipitation, and inhibition of sporozoite invasion. All three constructs were found to be immunogenic when administered alone, but antibody responses were greater for the larger constructs, R32tet32 and R48tet32. Increased dose, boosting, and the use of adjuvants further augmented antibody responses. R32tet32 was found to be the most immunogenic of the three constructs, and high levels of protective antibodies were found to persist for at least 44 weeks when the construct was given with alum. Clinical trials with alum adjuvanted R32tet32 have now begun.     

Two-step chromatographic purification of recombinant Plasmodium falciparum circumsporozoite protein from Escherichia coli

The Plasmodium falciparum circumsporozoite (PfCS) protein (aa 19–405) has been cloned and expressed in E. coli. The protein was purified in a two-step process that was rapid and reproducible. E. coli cells were grown to a high density before induction for 1 h. Cells were disrupted by high pressure microfluidization and the total bacterial protein solubilized in 6 M Gu-HCl. The protein was refolded while bound to Ni–NTA agarose by exchange of 6 M Gu-HCl for 8 M urea and then slow removal of the urea. The eluted protein was further purified on Q Sepharose Fast Flow using conditions developed to remove E. coli proteins and reduce endotoxin (to 10 EU/50 μg). Yield was 20 mg of PfCS protein from 10 g of wet cell paste. The final protein product bound to HepG2 liver cells in culture and inhibited the invasion of those cells by sporozoites in an ISI assay greater than 80% over control cultures when used at 10 μg/ml.     

Plasmodium falciparum: restricted polymorphism of T cell epitopes of the circumsporozoite protein in Brazil.

We examined the extent of variation of the 3' region of the circumsporozoite gene among Plasmodium falciparum isolates through amplification of a selected DNA fragment followed by DNA sequencing. A total of 32 isolates were analyzed, of which 24 were from Amazon endemic areas in Brazil and 8 from widely separated geographical regions in the world. Among Brazilian isolates only 2 variants were detected: 19 displayed the same sequence of strain 7G8 whereas the 4 remaining isolates differed from the 7G8 strain at five nucleotide positions which also led to amino acid changes. Variation was restricted to one of the T-helper epitopes while the sequence identified as a cytotoxic T cell epitope was conserved in all Brazilian isolates. P. falciparum samples from other geographical regions in the world showed sequences distinct from those of Brazilian isolates. However, some constancy could be observed within that variation. For instance, the most frequent nucleotide substitutions, from A and C at nucleotide positions 1015 and 1024, were the same in all isolates.     

Sequence variation in putative functional domains of the circumsporozoite protein of Plasmodium falciparum. Implications for vaccine development

Sequences of the circumsporozoite protein gene from five isolates of the human malaria parasite Plasmodium falciparum are compared, and the extent of sequence variability within putative functional domains is assessed in terms relating to vaccine efficacy. Nucleotide substitutions were observed outside of the immunodominant domain. Of the substitutions observed outside of the repeat domain, none were silent. The substitutions correlated with biologically functional regions, such as a helper T cell epitope (Th2R) and a region (N1) which may be important in liver invasion. Contrary to previous impressions, the small numbers of amino acid changes in these areas of the protein seem potentially very significant. The immunodominant repeat region displays several characteristics that implicate a rapid evolutionary mechanism, most probably involving recombination. The data supporting this are 1) variable numbers of repeats, 2) a shifting pattern of substitutions among the isolates, and 3) codon bias. The region thus has the potential for very rapid change should an effective anti-repeat vaccine come into use. We conclude that strain variability is significant, that the potential for large scale variation in the repeats is great, and that regions that may be critical for an effective vaccine are polymorphic. Their potential impact on malaria vaccine development must be addressed.     

Evolution of the immunodominant domain of the circumsporozoite protein gene from Plasmodium vivax. Implications for vaccines

Recent work directed toward the development of a malarial vaccine has focused on the identification and production of the immunodominant repeating peptide of the circumsporozoite protein of the human malaria parasites as an antigen. An important factor which relates to the usefulness of this antigen in a vaccine is the rate at which the molecule changes in sequence. We have determined the sequence and arrangement of the repeating epitope of the circumsporozoite protein gene from a Plasmodium vivax isolate from La Paz, El Salvador (Sal-I). This is compared with a portion of the previously published sequence of the circumsporozoite protein gene from a P. vivax isolate from Belém, Brazil. The genes appear to be very similar in the repeat region. There are 20 similar repeating units in the El Salvador strain and only 19 units are conserved in the Brazilian strain. Following this there are degenerate repeats in both strains. Even the pattern of silent mutations in the repeat area are similar; however, they are not necessarily in the identical location and appear to have shifted. The data suggest that the repeat region of these genes may be evolving by an accelerated mechanism(s). Such a phenomenon could severely decrease the long-term efficacy of a repeat-based anti-sporozoite vaccine.     

A role for apical membrane antigen 1 during invasion of hepatocytes by Plasmodium falciparum sporozoites

Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and invade hepatocytes as a first and obligatory step of the parasite life cycle in man. Hepatocyte invasion involves proteins secreted from parasite vesicles called micronemes, the most characterized being the thrombospondin-related adhesive protein (TRAP). Here we investigated the expression and function of another microneme protein recently identified in Plasmodium falciparum sporozoites, apical membrane antigen 1 (AMA-1). P. falciparum AMA-1 is expressed in sporozoites and is lost after invasion of hepatocytes, and anti-AMA-1 antibodies inhibit sporozoite invasion, suggesting that the protein is involved during invasion of hepatocytes. As observed with TRAP, AMA-1 is initially mostly sequestered within the sporozoite. Upon microneme exocytosis, AMA-1 and TRAP relocate to the sporozoite surface, where they are proteolytically cleaved, resulting in the shedding of soluble fragments. A subset of serine protease inhibitors blocks the processing and shedding of both AMA-1 and TRAP and inhibits sporozoite infectivity, suggesting that interfering with sporozoite proteolytic processing may constitute a valuable strategy to prevent hepatocyte infection.     

Antigenic analysis of the repeat domain of the circumsporozoite protein of Plasmodium vivax

In the present study we analyzed the fine specificity of mouse monoclonal and human polyclonal antibodies directed against the repeat domain of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium vivax. Five synthetic peptides, representing monomeric and dimeric repeats of this malarial antigen, were assayed for their capacity to inhibit the binding of these antibodies to a yeast-derived recombinant CS protein. The results revealed the existence of at least two distinct repeated overlapping epitopes in the CS protein of P. vivax. Furthermore, polyclonal sera contain antibodies which recognize additional determinants not represented by the synthetic repeat peptides. Some of these sera contain antibodies recognizing a region flanking the repeat domain (region I). The present findings are in contrast with the antibody response in rodents and humans to the Plasmodium falciparum CS protein, which is directed against a single repeated immunodominant epitope.