Sporozoite Stage Research Articles

Vaccines for preventing malaria (pre-erythrocytic)

Vaccines against all stages of the malaria parasite are in development, mainly for Plasmodium falciparum, which causes the most serious form of malaria. Pre-erythrocytic vaccines act to prevent or delay a malaria attack by attacking the sporozoite and liver stages before the parasite reaches the bloodstream. To assess the efficacy and safety of pre-erythrocytic malaria vaccines against any type of human malaria. In March 2006, we searched the Cochrane Infectious Diseases Group Specialized Register, CENTRAL (The Cochrane Library 2006, Issue 1), MEDLINE, EMBASE, LILACS, and the Science Citation Index. We also searched conference proceedings and reference lists of articles, and contacted organizations and researchers in the field. Randomized controlled trials comparing pre-erythrocytic vaccines with placebo, control vaccine, or routine antimalarial control measures in people of any age receiving an artificial challenge or natural exposure to malaria infection. Both authors independently assessed trial quality and extracted data. Results of meta-analyses were expressed as relative risks with 95% confidence intervals (CI) using an intention-to-treat analysis. Nine safety and efficacy trials, and two safety trials, with over 3000 participants were included. In semi-immune children, RTS,S vaccine reduced clinical episodes of malaria by 26% (95% CI 13% to 37%) and severe malaria by 58% (95% CI 15% to 79%) for up to 18 months. Prevalence of parasitaemia was also reduced by 26% (95% CI 11% to 38%) at six months after immunization. RTS,S also reduced clinical malaria episodes by 63% (95% CI 18% to 83%) in semi-immune adult men in the second year of follow up after a booster dose. No severe adverse events were judged to be related to RTS,S vaccine, although the frequencies of injection site pain, swelling, arm motion limitation, headache, and malaise were increased in the vaccine groups. There was no evidence for effect of the CS-NANP vaccines (307 participants, 3 trials), CS102 peptide vaccine (14 participants, 1 trial), or the ME-TRAP vaccine (372 participants, 1 trial). RTS,S vaccine was effective in preventing a significant number of clinical malaria episodes, including good protection against severe malaria in children for 18 months. No severe adverse events were attributable to the vaccine. Progression of this vaccine towards licensing is justified while efforts to increase its efficacy continue. The other vaccines do not look promising and further research is a priority.

Current status of vaccine development againstTheileriaparasites

The tick-borne protozoan parasites Theileria parva and Theileria annulata cause economically important diseases of cattle in tropical and sub-tropical regions. Because of shortcomings in disease control measures based on therapy and tick control, there is a demand for effective vaccines against these diseases. Vaccines using live parasites have been available for over two decades, but despite their undoubted efficacy they have not been used on a large scale. Lack of infrastructure for vaccine production and distribution, as well as concerns about the introduction of vaccine parasite strains into local tick populations have curtailed the use of these vaccines. More recently, research has focused on the development of subunit vaccines. Studies of immune responses to different stages of the parasites have yielded immunological probes that have been used to identify candidate vaccine antigens. Immunisation of cattle with antigens expressed in the sporozoite, schizont or merozoite stages has resulted in varying degrees of protection against challenge. Although the levels of protection achieved have not been sufficient to allow exploitation for vaccination, there are clearly further lines of investigation, relating to both the choice of antigens and the antigen delivery systems employed, that need to be pursued to fully explore the potential of the candidate vaccines. Improved knowledge of the molecular biology and immunology of the parasites gained during the course of these studies has also opened up opportunities to refine and improve the quality of live vaccines.

Eimeria tenella enolase and pyruvate kinase: A likely role in glycolysis and in others functions

Two cDNA codings for glycolytic enzymes were cloned from a cDNA library constructed from the schizont stage of the avian parasite Eimeria tenella. Enolase and pyruvate kinase cDNA were fully sequenced and compared with sequences of enzymes from other organisms. Although these enzymes were already detected in the sporozoite stage, their expression was enhanced during the first schizogony in accordance with the anaerobic conditions of this part of the life cycle of the parasite. Under activating conditions, microscopic observations suggest that these glycolytic enzymes were relocalised inside sporozoites and moreover were in part secreted. The enzymes were also localised at the apex of the first generation of merozoites. Enolase was partly observed inside the nucleus of sporozoites and schizonts. Taken together, these results suggest that glycolytic enzymes not only have a function in glycolysis during anaerobic intracellular stages but may also participate in the invasion process and, for enolase, in the control of gene regulation.

Differential evidence of natural selection on two leading sporozoite stage malaria vaccine candidate antigens

Experimental malaria vaccines based on two sporozoite stage candidate antigens of Plasmodium falciparum, the circumsporozoite protein (CSP) and thrombospondin-related adhesive protein (TRAP), have undergone clinical trials of efficacy. The relevance of naturally existing polymorphism in these molecules remains unknown. Sequence polymorphism in the genes encoding these antigens was studied in a Gambian population (sample of 48 trap and 44 csp gene sequences) to test for signatures of selection that would result from naturally acquired immunity. Allele frequency distributions were analyzed and compared with data from another population (in Thailand). Patterns of non-synonymous and synonymous polymorphism in P. falciparum and in Plasmodium vivax were compared with divergence from related species. Results indicate that polymorphism in TRAP is under strong selection for amino acid sequence diversity and that allele frequencies are under balancing selection within the Gambian P. falciparum population. There was no such evidence for CSP, calling into question the idea that most polymorphisms in this gene are under immune selection. There was a weak trend for regions known to encode T cell epitopes to have slightly higher indices suggesting balancing selection. Overall, the results predict more allele-specific immunity to TRAP than to CSP and should be considered in design and efficacy testing of vaccine candidates based on these antigens.

Quantitative imaging of Plasmodium sporozoites in the mammalian host

Malaria, the disease caused by Plasmodium, kills more than 1 million people annually. Little is known of the pre-erythrocytic phase of the parasite life cycle, i.e., after the sporozoite stage is inoculated in the dermis by a mosquito and before the erythrocyte-infecting stage is released from hepatocytes. We present here a quantitative, real-time analysis of the fate of parasites transmitted in a rodent system. We describe previously unrecognized steps in the parasite's journey to the liver of the host, which are likely to play an important role in the host immune response. To cite this article: R. Amino et al., C. R. Biologies 329 (2006).

A ubiquitous Plasmodium protein displays a unique surface labeling pattern in sporozoites

The Plasmodium sporozoite is infective for mosquito salivary glands and vertebrate host tissues. Although it is a key developmental stage of the malaria parasite, relatively few sporozoite surface or secreted proteins have been identified and characterized. Herein, we describe the molecular and cellular characterization of a novel surface molecule that is preferentially-expressed in salivary gland sporozoites as compared to oocyst and hemolymph sporozoites. This molecule, designated the sporozoite and erythrocytic stages (SES) protein (formerly known as Pg4), exhibits a spiral surface labeling pattern that spans over a known sporozoite surface antigen, the circumsporozoite protein, with only minor co-localization. SES consists of 551 amino acids encoding a putative 63.2 kDa protein that has been shown to be expressed not only on particular sporozoite stages, but also during the asexual and gametocyte stages. This novel protein also has three domains of unknown function that are conserved in at least eight Plasmodium spp. that represent human, avian, non-human primate, and rodent malarias.

Vaccines for preventing malaria (SPf66).

A malaria vaccine is badly needed. SPf66 was one of the earliest vaccines developed. It is a synthetic peptide vaccine containing antigens from the blood stages of malaria linked together with an antigen from the sporozoite stage, and is targeted mainly against the blood (asexual) stages. To assess the effect of SPf66 malaria vaccines against Plasmodium falciparum, P. vivax, P. malariae, and P. ovale in preventing infection, disease, and death. We searched the Cochrane Infectious Diseases Group Specialized Register (September 2005), CENTRAL (The Cochrane Library 2005, Issue 3), MEDLINE (1966 to September 2005), EMBASE (1980 to September 2005), LILACS (1982 to September 2005), Science Citation Index (1981 to September 2005), and reference lists of articles. We also contacted organizations and researchers in the field. Randomized and quasi-randomized controlled trials comparing SPf66 vaccine with placebo or routine antimalarial control measures in people of any age receiving an artificial challenge or natural exposure to malaria infection (any species). Two people independently assessed trial quality and extracted data, including adverse events. Results were expressed as relative risks (RR) with 95% confidence intervals (CI). Ten efficacy trials of SPf66 involving 9698 participants were included. Results with SPf66 in reducing new episodes of P. falciparum malaria were heterogeneous: it was not effective in four African trials (RR 0.98, 95% CI 0.90 to 1.07; 2371 participants) or in one Asian trial (RR 1.06, 95% CI 0.90 to 1.25; 1221 participants). In four trials in South America the number of first attacks with P. falciparum was reduced by 28% (RR 0.72, 95% CI 0.63 to 0.82; 3807 participants). It did not reduce episodes of P. vivax malaria or admission to hospital with severe malaria. Trials have not indicated any serious adverse events with SPf66 vaccine. There is no evidence for protection by SPf66 vaccines against P. falciparum in Africa. There is a modest reduction in attacks of P. falciparum malaria following vaccination with SPf66 in South America. There is no justification for further trials of SPf66 in its current formulation. Further research with SPf66 vaccines in South America or with new formulations of SPf66 may be justified.

Japanese Babesia microti Cytologically Detected in Salivary Glands of Naturally Infected Tick Ixodes ovatus

Babesia microti protozoa were detected by light and electron microscopy in the salivary glands of field-collected Ixodes ovatus ticks; 6 of 85 adult ticks were demonstrated to be positive for B. microti DNA by polymerase chain reaction assays. In the salivary glands of unfed ticks, B. microti existed in the sporoblast stage in the granular acinus cells, and developed into the sporozoite stage during feeding on the host for 2 days. The present results indicated for the first time that I. ovatus can indeed carry B. microti and is not infected mechanically with the parasites by blood-sucking. This frequent infection of I. ovatus with B. microti demonstrates the significance of such a vector-pathogen relationship in Japan, and strongly suggests that I. ovatus is involved in the maintenance of B. microti in the fauna of Japanese rodents.

MOSQUITO SALIVARY GLANDS LURE MALARIA PARASITES

James Dvorak has devoted his career to the study of malaria, `one of the major maladies of our time.' Over the years, Dvorak has probed the intricate details of the malaria parasite's voyage inside its mosquito host. Now,working with Mayumi Akaki, Dvorak has found that the sporozoite stage of the malaria parasite's lifecycle finds the chemical lure of mosquito salivary glands irresistible (p. 3211).The malaria parasite divides its lifecycle between two hosts: humans and mosquitoes. When a mosquito gorges on a blood meal from a person infected with malaria, it unwittingly sucks the parasite's gametocytes into its body. Once inside the mosquito, the gametocytes fuse and the resulting ookinete invades the mosquito's gut wall. Eventually, the developing oocyte bursts, releasing delicate spindle-shaped sporozoites into the insect's circulatory system. Coursing around the mosquito's body, the sporozoites are incredibly selective;they only invade the insect's salivary glands, ready for the mosquito to feast on its next human victim.Dvorak wanted to know how sporozoites make their way to a mosquito's salivary glands. `This is a tricky question to answer', he says, explaining that the salivary glands are tucked away in a mosquito's thorax, which is covered by an opaque cuticle that makes it impossible to study the glands directly under a microscope. Dvorak suspected that sporozoites use a chemical cue to locate the salivary glands as they are swept along by the mosquito's circulatory system. Teaming up with Akaki, he painstakingly developed an in vitro system to discover whether sporozoites are attracted to mosquito salivary glands.First, Dvorak and Akaki needed to show that sporozoites can steer themselves in a particular direction. If you place the parasites on a glass slide and peer down a microscope, you'll see that the sporozoites glide around in elegant circles. `But that doesn't get us anywhere, and it certainly doesn't get the sporozoites anywhere!' Dvorak remarks. To find out whether the parasites were simply being constrained by the 2D environment, Dvorak and Akaki created 3D surroundings that would allow the parasites to show off their locomotor skills. They developed a tiny chamber that holds a microscopic amount of Matrigel, a semi-solid matrix, and loaded the chamber with green fluorescent protein-expressing sporozoites. Capturing every move of the fluorescent parasites using a video camera and a 3D motion-tracking program,they were delighted to see that the sporozoites adeptly travelled through the Matrigel using a corkscrew-like motion.But are the sporozoites attracted to mosquito salivary glands? To find out,Dvorak and Akaki added salivary gland extract to one end of the chamber. Sure enough, the sporozoites navigated towards the extract. After carefully ruling out other explanations for the sporozoites' movements, the pair concluded that the parasites use chemotactic gradients to locate a mosquito's salivary glands.Dvorak and Akaki's findings offer a tantalising new approach in the fight against malaria. Treating a mosquito with an anti-chemotactic substance to counteract the sporozoites' chemical attraction to the salivary glands could leave the parasites stranded without a map on their long journey through the mosquito body.

Malaria held captive

Malaria sporozoites that lack a certain protease get trapped in their cyst-like breeding ground (the oocyst), as shown by Aly and Matuschewski (page 225). Unable to break free, these captive parasites turn in helpless circles inside the oocyst and their life cycle halts. Figure Malaria sporozoites that lack egress cysteine protease 1 get trapped inside the oocyst (shown) in the mosquito midgut. Malaria-causing parasites (Plasmodium species) have a complex life cycle that includes several distinct stages of intracellular growth in vertebrate and mosquito hosts. Movement from one stage to the next requires exit from one host cell and entry into another. Invasion of target cells has been shown to require parasite-encoded proteases that, by poorly defined mechanisms, allow the bug to penetrate cell membranes. Parasite exit from target cells has been even less well-studied.Aly and Matuschewski now identify a cysteine protease that is selectively expressed at the late sporozoite stage, just before sporozoite release and entry into the mosquito salivary glands. Elimination of this protease from the parasite caused mature sporozoites to become trapped inside the oocyst, a protective compartment forged from the basal lamina of the mosquito midgut where the sporozoites divide and grow. There they commenced an unusual circular motion, the significance of which is not yet understood. These data suggest that Plasmodium egress is an active process that requires the protease—dubbed egress cysteine protease 1—to actively break down the oocyst wall, rather than a passive one in which the oocyst simply ruptures once it is filled to capacity.

Correction: Immune responses toPlasmodium vivax pre-erythrocytic stage antigens in naturally exposed Duffy-negative humans: a potential model for identification of liver-stage antigens (Vol. 35(6) 2005, DOI 10.1002/eji.200425807)

Unfortunately, in this article (in this issue) the labels indicating the different groups in column “Donors” are missing in the published Fig. 4. The complete correct Fig. 4 is as given here: IFN-γ responses to P. vivax proteins in humans with different Duffy phenotypes. Frozen PBMC from Fy(–) P. vivax exposed individuals, Fy(+) P. vivax exposed individuals, or unexposed controls were assayed for IFN-γ induction by ELISPOT after stimulation with autologous B or M-DC transfected with novel P. vivax proteins. As positive controls, IFN-γ responses in Fy(–) individuals were detected preferentially to known P. vivax sporozoite Ag (CSP or SSP2) but not to blood stage Ag (MSP-1, AMA-1, or DBP), whereas, the responses in Fy(+) humans were detected to both sporozoite and blood stage Ag. IFN-γ responses to eight novel P. vivax proteins were detected in P. vivax exposed individuals, but not in unexposed controls. IFN-γ responses to three of the eight responded P. vivax Ag (PVA9, PVA20 and PVA22) were detected only in Fy(–) but not Fy(+) individuals who were exposed to P. vivax, whereas the responses to other five Ag (PVA3, PVA16, PVA18, PVA19, PVA21) were detected in both Fy(–) and Fy(+)P. vivax exposed individuals. IFN-γ responses to PVA6, PVA13 and PVA17 were detected in 18 of the 19 donors tested including P. vivax exposed and unexposed controls, indicating these three Ag are cross-reactive T cell Ag; a the numbers in the black boxes indicate the number of Ag-specific IFN-γ-producing cells/million PBMCs;b based on proteomics analysis by Florens et al. [16]; spz, sporozoite; mrz, merozoite; tpz, trophozoite; gmt, gametocyte. See original article http://dx.doi.org/10.1002/eji.200425807

Amino acid sequence constraint and gene expression pattern across the life history in the malaria parasite Plasmodium falciparum

The relationship between gene expression across the life cycle and protein conservation in Plasmodium falciparum was examined by comparing gene expression data for six life-history stages with the number of nonsynonymous substitutions per site ( d N) between 901 orthologous gene pairs of P. falciparum and Plasmodium yoelii. A high level of expression across the life history was associated with decreased d N and thus with protein conservation. By contrast, differential expression in the sporozoite and merozoite stages was associated with increased d N. At least some sporozoite- and merozoite-expressed genes with high d N have probably been subject to positive selection arising from parasite–host coevolution. A high level of expression across the life history was associated with higher than average G + C content at the first and second codon positions, whereas a high level of expression in the sporozoite and merozoite was associated with reduced G + C content at the first and second codon positions, the latter pattern evidently reflecting the relaxation of constraint on the amino acid sequence.

Paleohaemoproteus burmacisgen. n., sp. n. (Haemospororida: Plasmodiidae) from an Early Cretaceous biting midge (Diptera: Ceratopogonidae)

Paleohaemoproteus burmacis gen. n., sp. n. (Haemospororida: Plasmodiidae) is described from the abdominal cavity of a female biting midge (Diptera: Ceratopogonidae) preserved in 100 million year old amber from Myanmar (Burma). The description is based on the developmental stages of oocysts and sporozoites. The fossil species differs from extant species of Haemoproteus by its wide range of oocyst sizes, small sporozoites and occurrence in an extinct species of biting midge. Numerous sporozoites in the abdominal cavity suggest that the biting midge was an effective vector of this malarial parasite. Characters of the biting midge suggest that the host was a large, cold-blooded vertebrate. This is the earliest record of a malaria parasite and first indication that Early Cretaceous reptiles were infected with haemosporidial parasites.

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.

High-throughput generation of P. falciparum functional molecules by recombinational cloning.

Large-scale functional genomics studies for malaria vaccine and drug development will depend on the generation of molecular tools to study protein expression. We examined the feasibility of a high-throughput cloning approach using the Gateway system to create a large set of expression clones encoding Plasmodium falciparum single-exon genes. Master clones and their ORFs were transferred en masse to multiple expression vectors. Target genes (n = 303) were selected using specific sets of criteria, including stage expression and secondary structure. Upon screening four colonies per capture reaction, we achieved 84% cloning efficiency. The genes were subcloned in parallel into three expression vectors: a DNA vaccine vector and two protein expression vectors. These transfers yielded a 100% success rate without any observed recombination based on single colony screening. The functional expression of 95 genes was evaluated in mice with DNA vaccine constructs to generate antibody against various stages of the parasite. From these, 19 induced antibody titers against the erythrocytic stages and three against sporozoite stages. We have overcome the potential limitation of producing large P. falciparum clone sets in multiple expression vectors. This approach represents a powerful technique for the production of molecular reagents for genome-wide functional analysis of the P. falciparum genome and will provide for a resource for the malaria resource community distributed through public repositories.

Molecular genetic characterization and subcellular localization of a putative Theileria annulata membrane protein

A Theileria annulata protein (TaD) exhibiting an N-terminal signal sequence for endoplasmic reticulum membrane translocation and a conserved cysteine-rich region was isolated by screening the mRNA of a T. annulata-infected bovine lymphoblastoid cell line with degenerated primers directed against T. annulata-targeting sequences. The TaD-coding sequence was found to be most closely related to the genomic DNA sequence of T. parva (TIGR database, 72%) and the amino acid sequence of Plasmodium falciparum (41%), P. yoelii yoelii (38%) and Cryptosporidium parvum (36%). The TaD mRNA is expressed within the sporozoite, schizont and merozoite stages of the parasite, implying that it is constitutively transcribed throughout the parasite's life cycle. Allelic variants were found between isolates originating from different geographical regions, however not affecting conserved cysteines. The open reading frame encoded a protein of 19.5 kDa and non-reducing SDS-PAGE analysis demonstrated a homodimeric protein. Using confocal microscopy, the protein was found to be both located in the parasite cytoplasm and to colocalize with a transmembrane protein of the schizonts within infected cells.

Babesia bigemina: Sporozoite Isolation from Boophilus microplus Nymphs and Initial Immunomolecular Characterization

It has been hypothesized that babesial sporozoites express specific antigens that induce protective immunologic responses in cattle. However, they remain uncharacterized, partly for lack of research on the sporozoite stage of Babesia spp. This field suffers from complete knowledge of parasite development in the tick salivary gland; limited amounts of sporozoites from ticks, and a lack of protocols for induction and purification of sporozoites. In this work, Boophilus microplus larvae infected with B. bigemina were fed on susceptible cattle. Nymphs were collected and macerates were separated by a Percoll density gradient. Microscopic analysis of Giemsa-stained smears showed a larger number of sporozoites from nymphs fed for 9 days. Percoll-purified sporozoites were observed in large numbers in groups or individually and free of tick cells. RT-PCR analysis of total RNA extracted from purified sporozoites indicated transcription of the rhoptry associate protein 1 (rap-1) genes: rap-1a, rap-b, rap-1c, as well as the heat shock protein 20 (hsp-20) gene. Purified sporozoites were cultured in vitro analyzed for RAP-1a expression using an immunocytochemistry assay. Erythrocyte-attached sporozoites reacted with a specific RAP-1a monoclonal antibody. This is the first report of Babesia bigemina sporozoite antigens. Moreover, purified sporozoites will allow the characterization of stage-specific antigens involved in immunologic protection.

Cryptosporidium parvum at different developmental stages modulates host cell apoptosis in vitro.

We studied apoptosis in a human ileocecal adenocarcinoma tumor cell line (HCT-8) infected with Cryptosporidium parvum, from 2 to 72 h postinfection (h.p.i.). At 2 h.p.i., the percentage of annexin V-positive cells in the cell culture had increased to 10% compared to 2.5% in noninfected control culture; sorted infected cells expressed mRNA of FasL, the active form of caspase 3, and high caspase 3 activity, whereas the noninfected neighboring cells sorted from the same culture showed no signs of apoptosis. At 24 h.p.i., the percentages of early (annexin V positive) and late (DNA fragment) apoptotic cells were 13 and 2%, respectively, in the entire cell culture, and these percentages were not statistically significant in comparison with those from noninfected control cultures. At this time, sorted infected cells expressed the inactive form of caspase 3, a low caspase 3 activity, and the antiapoptotic protein Bcl-2. Noninfected cells sorted from the same culture showed expression of the active form of caspase 3, a moderate caspase 3 activity, and no Bcl-2 expression. At 48 h.p.i., the percentages of early and late apoptotic cells and caspase 3 activity had increased in the total cell culture, and both sorted infected and noninfected cells showed the active form of caspase 3. These results show that C. parvum, depending on its developmental stage, can inhibit (at the trophozoite stage) or promote (at the sporozoite and merozoite stages) host cell apoptosis, suggesting that it is able to interact with and regulate the host-cell gene expression.

Towards a reference map of Eimeria tenella sporozoite proteins by two-dimensional electrophoresis and mass spectrometry

Eimeria tenella is a parasite of great importance as a disease causing agent in the poultry industry. Until recently, biological studies have focused on specific proteins, some of which play an important role in the parasite life cycle. Post-genomic studies will make it possible to understand the complexity of the parasites and their interactions with host cells. Here we present a systematic reference map of the proteins from E. tenella sporozoites. The proteins expressed at the sporozoite stage were resolved between isoelectric points 3–10 and 4–7. They were systematically identified using mass spectrometry and 16 known Eimeria sporozoite proteins were identified on two-dimensional maps. Peptide fragmentation data from mass spectrometry were compared to single and consensus expression sequence tags in databases and to the E. tenella genome (not annotated). Among the set of unknown proteins analysed, 12 new assignments were proposed on the basis of similarities with Apicomplexa proteins. In order to define sporozoite proteins as potential targets for coccidiosis therapy, proteins were studied according to their relative abundance and immunogenicity in the sporozoite. Immunoblots of sporozoite 2D maps with chicken sera were performed and approximately 50 proteins were defined as antigens. It was shown that abundance and immunogenicity are not related in the sporozoite stage. Perspectives of gene prediction and completion of the genome annotation by a proteomic approach is discussed.

Long-term multiepitopic cytotoxic-T-lymphocyte responses induced in chimpanzees by combinations of Plasmodium falciparum liver-stage peptides and lipopeptides.

Preclinical immunogenicity studies of 12 malaria peptides, selected from four Plasmodium falciparum antigens (Ags), namely, LSA1, LSA3, SALSA, and STARP, that are expressed at the pre-erythrocytic (sporozoite and liver) stages of the human parasite were carried out in chimpanzees. To strengthen their immunogenicity, six of these synthetic peptides were modified by the C-terminal addition of a single palmitoyl chain (lipopeptides) and delivered without adjuvant, whereas the remaining six unmodified peptides were emulsified and delivered by using Montanide ISA51 adjuvant. We have previously reported that these peptides and lipopeptides induce high B-cell and CD4(+)-T-helper responses in chimpanzees. In this report, we show their ability to induce multiepitopic and long-lasting antigen-specific CD8(+) cytotoxic-T-lymphocyte (CTL) responses. The magnitude, consistency, and memory of CTL responses generated by LSA3 peptides point to the strong immunogenicity of this liver-stage Ag. These findings support the screening strategy used to select the four P. falciparum pre-erythrocytic Ags and emphasize their valuable immunogenic properties. The successful immunization of nonhuman primates with combinations of corresponding peptides in a mineral oil emulsion (ISA51) and lipopeptides in saline provide a vaccine formulation that can be tested in humans.