Plasmodium Falciparum Sporozoites Research Articles

Biology of malarial liver stages: implications for vaccine design.

The molecular events controlling sporozoite invasion and exo-erythrocytic (EE) development within hepatocytes are largely not understood, and EE parasites are probably better defined immunologically than biologically. The observation that the Plasmodium falciparum sporozoite antigen TRAP (thrombospondin-related anonymous protein) contains multiple adhesive domains that recognize endothelial and hepatocyte receptors indicates that, like leucocyte passage across capillaries, sporozoite invasion probably involves a co-ordinated interaction between sporozoite and hepatic molecules. The parallel with leucocyte extravasation is strengthened by the finding that TRAP contains a functional, integrin-like, I domain. EE parasites are an important target of immunity elicited by irradiated sporozoites, and much current effort is focused on developing malaria vaccines targeting EE parasites. Only one EE-specific antigen, liver-stage antigen 1 (LSA-1), is known to be expressed during EE development and may contribute to protective immunity elicited by irradiated P. falciparum sporozoites. In a study in Papua New Guinea, resistance to P. falciparum infection correlated with CD8+ T-cell interferon-gamma responses to an LSA-1 epitope that contains an HLA A11-restricted sequence. Since A11 is > 40% frequent in this population it is reasonable to suggest that, as with B53 responses to LSA-1 in The Gambia, P. falciparum has driven genetic selection of certain HLA haplotypes, as proposed by Haldane nearly 50 years ago. LSA-1 is thus an important vaccine candidate, and is being expressed in bacterial and phage vectors.

Midgut bacteria in Anopheles gambiae and An. funestus (Diptera: Culicidae) from Kenya and Mali.

Field studies in Kenya and Mali investigated the prevalence of bacteria in the midguts of malaria vectors, and the potential relationship between gram-negative bacteria species and Plasmodium falciparum sporozoites. Midguts were dissected from 2,430 mosquitoes: 863 Anopheles funestus Giles and 1,037 An. gambiae s.l. Giles from Kenya, and 530 An. gambiae s.l. from Mali. An. funestus had a higher prevalence of gram-negative bacteria (28.5%) compared with An. gambiae collected in Kenya and Mali (15.4 and 12.5%, respectively). Twenty different genera of bacteria were identified by gas chromatography from 73 bacterial isolates from mosquito midguts. Pantoea agglomerans (Enterobacter agglomerans) was the most common species identified. There was no association between gram-negative bacteria in the midgut and P. falciparum sporozoites in field-collected An. gambiae s.l. and An. funestus. However, An. funestus females that harbored gram positive bacteria were more likely to be infected with sporozoites compared with those with no cultivable bacteria or gram negative bacteria in their midguts. Habitat-related variation in the prevalence of diverse types of bacteria in mosquitoes could influence malaria parasite development in mosquitoes and corresponding sporozoite prevalence.

Identification of human liver carboxylesterase as one of the proteins involved in Plasmodium falciparum malaria sporozoite invasion in primary cultures of human hepatocytes

Background/Aims: In a previous study, we have demonstrated that primary human hepatocytes in culture are susceptible for Plasmodium falciparum sporozoite invasion and for development of parasites into exoerythrocytic forms. In a separate study we demonstrated the involvement of two human liver plasma membrane proteins (55 kD and 20 kD) in the invasion of P. falciparum sporozoites in vitro. In this study we have unravelled the nature of the 55 kD protein. Methods: For the identification of this protein, a 53–58 kD membrane protein fraction from human liver was isolated, radioactively labelled, incubated with sporozoites and cross-linked. After reduction of the cross-linker, the released proteins were mixed with unlabelled 53–58 kD protein fraction and separated on two-dimensional SDS-PAGE. Autoradiography showed a single spot corresponding to a protein of 55 kD and pI of 5.7–5.8. Results: Amino acid sequencing revealed the 55 kD protein as carboxylesterase. The biological activity of purified human liver carboxylesterase and of an anti-serum against carboxylesterase on sporozoite invasion in vitro was evaluated. Human carboxylesterase as well as a rabbit antiserum against carboxylesterase inhibited the invasion of P. falciparum sporozoites into primary human hepatocytes in culture. A number of carboxylesterase cDNA clones were isolated from a human liver cDNA library. Sequence analysis revealed two different iso-types. Immunoaffinity purified recombinant human carboxylesterase was shown also to inhibit the invasion of sporozoites into primary human hepatocytes. Immunocytochemical analysis of the localisation of carboxylesterase in primary cultures of human hepatocytes using specific antibodies, showed its presence inside the hepatocytes and on the membrane. Conclusions: Carboxylesterase plays a role in the invasion process of P. falciparum sporozoites into human hepatocytes in vitro. The implications of these findings are further discussed.

Development of two monoclonal antibodies against Plasmodium falciparum sporozoite surface protein 2 and mapping of B-cell epitopes.

The Plasmodium yoelii sporozoite surface protein 2 (PySSP2) is the target of protective cellular immunity. Cytotoxic T cells specific for the Plasmodium falciparum analog PfSSP2, also known as thrombospondin-related anonymous protein (TRAP), are induced in human volunteers immunized with irradiated sporozoites. PfSSP2 is an important candidate antigen for a multicomponent malaria vaccine. We generated and characterized three monoclonal antibodies (MAbs) specific for PfSSP2/TRAP. The MAbs PfSSP2.1 (immunoglobulin G1 [IgG1]), PfSSP2.2 (IgG2a), and PfSSP2.3 (IgM) were species specific and identified three distinct B-cell epitopes containing sequences DRYI, CHPSDGKC, and TRPHGR, respectively. PfSSP2.1 partially inhibited P. falciparum liver-stage parasite development in human hepatocyte cultures (42 and 86% in two experiments at 100 microg/ml). Mice immunized with vaccinia virus expressing full-length PfSSP2 protein produced antibodies to (DRYIPYSP)3, and humans living in malaria-endemic areas (Indonesia and Kenya), who have lifelong exposure and partial clinical immunity to malaria, had antibodies to both (DRYIPYSP)3 and (CHPSDGKCN)2. Mice immunized with multiple antigen peptides MAP4 (DRYIPYSP)3P2P30 and MAP4 (CHPSDGKCN)3P2P30 in TiterMax developed antibodies to sporozoites that partially inhibited sporozoite invasion of human hepatoma cells (39 to 71% at a serum dilution of 1:50 in three different experiments). The modest inhibitory activities of the MAbs and the polyclonal antibodies to PfSSP2/TRAP epitopes do not suggest that a single-component vaccine designed to induce antibodies against PfSSP2/TRAP will be protective. Nonetheless, the MAbs directed against PfSSP2, and the peptides recognized by these MAbs, will be essential reagents in the development of PfSSP2/TRAP as a component of a multivalent P. falciparum human malaria vaccine.

Detection of Plasmodium falciparum sporozoites in naturally infected anopheline species using a fluorescein-labelled DNA probe

A dot blot hybridization method was developed to detect Plasmodium falciparum sporozoites in naturally infected mosquitoes. A fluorescein-labelled oligomer was used as a probe. Initial non-specific hybridization was found to correlate with the presence of blood in the mosquitoes. This was eliminated by allowing digestion of the engorged blood by keeping the mosquitoes in cages for 48 h before processing. The limit of detection of the hybridization assay was estimated to be about 500 sporozoites. The assay was evaluated on 198 indoor resting blood fed female Anopheles gambiae s.l mosquitoes collected from three malaria hypo- and meso-endemic areas in Ethiopia. An application of the polymerase chain reaction (PCR) amplifying a fragment of the K1-14 gene of P. falciparum was used as a reference method. P. falciparum sporozoites were detected in four specimens (2%) by hybridization assay and by PCR alike. The results of this study indicate that the hybridization method can be potentially valuable in large scale epidemiological studies for detection of P. falciparum sporozoites in naturally infected anopheline species.

Humoral immune responses in Gambians to Pfs16, an immunodominant, Plasmodium falciparum integral membrane protein.

Naturally acquired humoral immune responses to Pfs16, an integral membrane protein expressed in Plasmodium falciparum gametocytes and sporozoites, were investigated in The Gambia. A high prevalence of antibodies to this molecule was detected by peptide ELISA. Ninety-three per cent of the people taking part in a survey at the end of the rainy season (November) had serum antibodies to one or more synthetic peptides spanning the sequence: 88% reacted with one particular peptide sequence (IMLIILSGIVGFKVK) whereas only one out of ten non-Gambians (taking anti-malarial prophylaxis with no history of infection) reacted with the peptide. Epitope mapping with mouse MoAbs has shown that this peptide is located on a part of the molecule differing from immunodominant regions of the molecule identified in a previous study in Papua New Guinea.

The impact of permethrin-impregnated bednets on malaria vectors of the Kenyan coast.

The effects of introducing permethrin-impregnated bednets on local populations of the malaria vector mosquitoes Anopheles funestus and the An.gambiae complex was monitored during a randomized controlled trial at Kilifi on the Kenyan coast. Pyrethrum spray collections: inside 762 households were conducted between May 1994 and April 1995 after the introduction of bednets in half of the study area. All-night human bait collections were performed in two zones (one control and one intervention) for two nights each month during the same period. PCR identifications of An.gambiae sensu lato showed that proportions of sibling species were An.gambiae sensu stricto > An.merus > An.arabiensis. Indoor-resting densities of An.gambiae s.l. and the proportion of engorged females decreased significantly in intervention zones as compared to control zones. However, the human blood index and Plasmodium falciparum sporozoite rate remained unaffected. Also vector parous rates were unaltered by the intervention, implying that survival rates of malaria vectors were not affected. The human-biting density of An.gambiae s.l., the predominant vector, was consistently higher in the intervention zone compared to the control zone, but showed 8% reduction compared to pre-intervention biting rates-versus 94% increase in the control zone. Bioassay, susceptibility and high-performance liquid chromatography results all indicated that the permethrin content applied to the nets was sufficient to maintain high mortality of susceptible vectors throughout the trial. Increased rates of early outdoor-biting, as opposed to indoor-biting later during the night, were behavioural or vector composition changes associated with this intervention, which would require further monitoring during control programmes employing insecticide-treated bednets.

A novel Plasmodium falciparum sporozoite and liver stage antigen (SALSA) defines major B, T helper, and CTL epitopes.

In the search for subunit vaccines that are able to induce the type of sterile, protective immunity achieved by irradiated sporozoites, there is increasing evidence that defense mechanisms directed at the intrahepatic stage and Ags expressed at this stage are critical. We have initiated a systematic search for such molecules and report here the identification and partial characterization of a novel Plasmodium falciparum gene encoding a 70-kDa protein, expressed in both sporozoite and liver stages (SALSA), with a vaccine potential that stems from its antigenic features. Antigenicity and immunogenicity studies were conducted in individuals exposed to malaria, in immunized mice, and in chimpanzees, using a recombinant protein and two synthetic peptides. Results show that the SALSA nonrepetitive sequence defines 1) major B cell epitopes, as shown by a high prevalence of Abs to each peptide in three African areas differing in their level of endemicity; 2) Th epitopes, as demonstrated by lymphoproliferation and IFN-gamma secretion in cells from the individuals from one of the low transmission areas, as well as helper effect upon Ab secretion in mice; and 3) epitopes for cytolytic lymphocytes, demonstrated in immunized and sporozoite-challenged chimpanzees, and associated with MHC class I leukocyte Ags. The latter are of particular importance, because this is the only part of the malaria life cycle in which the parasite is located in a cell expressing class I Ags and because CD8+ lymphocytes were found to be responsible for protection in experimental models.

Experimental Human Plasmodium Falciparum Infections: Longitudinal Analysis of Lymphocyte Responses with Particular Reference to γδ T Cells

The kinetics of the gamma delta T-cell response was analysed in the context of the overall haematological response in subjects experimentally infected with sporozoites of Plasmodium falciparum. Numbers of gamma delta and alpha beta T cells and NK cells declined markedly during infection to reach minimum values 12-13 days post-infection when the patients were ill. This decline commenced from the beginning of the erythrocytic cycle and well before parasites could be detected microscopically and clinical symptoms developed. Platelet numbers also declined. In vivo activation of gamma delta T cells was evident with sequential up-regulation of the activation markers CD69 and HLA-DR. gamma delta T cell numbers were highest after treatment with the majority being CD4-CD8-, HLA-DR+ and showing reduced CD45RA expression. Contrary to some published observations gamma delta T-cell percentages remained within the normal range. Little evidence of upregulation of activation or memory markers was observed in the alpha beta T-cell population. In vitro proliferative responses to malaria antigen which involve gamma delta T cells were lost as the infection progressed and the lymphocyte count declined but these could be restored with the addition of exogenous IL-2 to cultures. The authors findings are consistent with a protective and/or immunomodulatory role for gamma delta T cells in malaria.

Irradiated sporozoite vaccine induces HLA-B8-restricted cytotoxic T lymphocyte responses against two overlapping epitopes of the Plasmodium falciparum sporozoite surface protein 2.

Vaccines designed to protect against malaria by inducing CD8+ cytotoxic T lymphocytes (CTL) in individuals of diverse HLA backgrounds must contain multiple conserved epitopes from various preerythrocytic-stage antigens. Plasmodium falciparum sporozoite surface protein 2 (PfSSP2) is considered an important antigen for inclusion in such vaccines, because CD8+ CTL against the P. yoelii SSP2 protect mice against malaria by eliminating infected hepatocytes. To develop PfSSP2 as a component of malaria vaccines, we investigated the presence of anti-PfSSP2 CTL in two HLA-B8+ volunteers immunized with irradiated P. falciparum sporozoites and characterized their CTL responses using PfSSP2-derived 15-amino acid peptides bearing the HLA-B8-binding motif. Peripheral blood mononuclear cells from both volunteers stimulated with recombinant vaccinia expressing PfSSP2 displayed antigen-specific, genetically restricted, CD8+ T cell-dependent CTL activity against autologous target cells expressing PfSSP2. Of the five HLA-B8 motif-bearing 15-mers identified in the PfSSP2 sequence, two peptides sharing a 10-amino acid overlap sensitized HLA-B8-matched target cells from both volunteers for lysis by peptide-stimulated effectors. The CTL activity was HLA-B8 restricted and dependent on CD8+ T cells. Analysis of the three shorter peptides representing HLA-B8 motif-bearing sequences within the two positive peptides for their ability to bind to HLA-B8 in vitro, and to sensitize target cells for lysis by effectors stimulated with the 15-mers, identified two overlapping HLA-B8-restricted CTL epitopes. Available data indicate that the sequence of one CTL epitope is conserved and the other is variant among P. falciparum isolates. Circulating activated CTL against the conserved epitope could be directly identified in one of the two volunteers. The identification of two HLA-B8-restricted CTL epitopes on PfSSP2 provides data critical to developing an epitope-based anti-liver stage malaria vaccine.

T lymphocytes from volunteers immunized with irradiated Plasmodium falciparum sporozoites recognize liver and blood stage malaria antigens.

The model of protective immunity induced by immunization with irradiated plasmodia sporozoites (SPZ) has become the prototype for a promising vaccine strategy based on Ab and CTL responses directed against pre-erythrocytic stage Ags, in particular the circumsporozoite protein (CSP) and sporozoite surface protein 2 (SSP2). However, results from recently conducted vaccine studies suggest that T cell responses directed against additional specificities might also be required for protection. We have tested this hypothesis by examining human T lymphocytes from irradiated Plasmodium falciparum SPZ-immune volunteers for proliferative reactivities to parasitized red blood cells (pRBC) and recombinant proteins and synthetic peptides representing certain liver and blood stage Ags. In this work, we report that although SPZ-induced protective immunity is stage-specific, SPZ-immune lymphocytes recognized determinants associated with erythrocytic and liver stage parasites. Thus, protective immunity induced by irradiated SPZ may depend upon responses against pre-erythrocytic Ags in addition to CSP and SSP2.

Detection of Plasmodium sporozoites in mosquitoes by polymerase chain reaction and oligonucleotide rDNA probe, without dissection of the salivary glands.

Dried Anopheles gambiae mosquito head+thorax portions, infected with Plasmodium falciparum sporozoites, were processed by the polymerase chain reaction. The PCR product was hybridized to an oligonucleotide probe (known as 114R or AW34) diagnostic for Plasmodium. The detection level by autoradiography was ten sporozoites per mosquito. Head+thorax of mosquitoes that contained mature P.falciparum oocysts, without sporozoites, gave no positive signal, indicating that the test detects only infective mosquitoes. This test can be applied to wild mosquito specimens collected, prepared and processed at different time intervals. The technique is convenient, highly sensitive, and could be used with a non-radioactive detection system and specific probes to differentiate Plasmodium spp.

HLA-A2-restricted cytotoxic T lymphocyte responses to multiple Plasmodium falciparum sporozoite surface protein 2 epitopes in sporozoite-immunized volunteers.

CD8+ CTL specific for the Plasmodium yoelii sporozoite surface protein 2 (PySSP2) protect mice against malaria. For this reason, vaccines designed to induce CTL against P. falciparum SSP2 (PfSSP2) are under development. Optimal development of PfSSP2 as a component of human malaria vaccines requires characterization of HLA class I-restricted CTL against this Ag. For this purpose, PBMC from four HLA-A2+ human volunteers immunized with P. falciparum irradiated sporozoites were stimulated with a recombinant vaccinia virus expressing PfSSP2 and with 35 PfSSP2-derived 15-amino acid peptides containing sequences conforming to HLA-A2 binding motifs. Ag-specific, genetically restricted, CD8+ T cell-dependent cytotoxic activity against autologous target cells transfected with the PfSSP2 gene was demonstrated in the four volunteers. Twelve of the 35 peptides sensitized HLA-A2-matched target cells for lysis by peptide-stimulated effectors. Three volunteers had CTL against 9 of the 12 peptides, and one had no peptide-specific CTL. HLA-A*0201 restriction was confirmed by demonstrating that effectors from the three responders could be stimulated with six different peptides to lyse HLA-A*0201+ T2 cells incubated with the homologous peptides. Peptide-specific, genetically restricted, CD8+ T cell-dependent cytotoxic activity was also demonstrated against two peptides using unstimulated PBMC as effectors. Available data indicate that the motif-bearing sequences in 6 of the 12 positive peptides are conserved among P. falciparum isolates and clones. Demonstration of HLA-A2-restricted CTL responses to multiple PfSSP2-derived peptides, and of circulating activated CTL against PfSSP2 in immune volunteers provide important information for optimal design and evaluation of vaccines containing this pre-erythrocytic stage Ag.

Induction of humoral immune response against Plasmodium falciparum sporozoites by immunization with a synthetic peptide mimotope whose sequence was derived from screening a filamentous phage epitope library

The mouse monoclonal antibody 2A10 (immunoglobulin G), which recognizes the (NANP)n repeat of Plasmodium falciparum circumsporozoite surface protein, was used to screen a filamentous phage epitope library expressing random amino acid hexamers. The sequences obtained were TNRNPQ, SNRNPQ, NND-NPQ, SNYNPQ, and QNDNPQ (single-letter amino acid designation). These peptides showed 50% homology with the native epitope (PNANPN) and therefore were considered to mimic its structure (mimotopes). Two of these mimotopes (TNRNPQ and NNDNPQ) inhibited the binding of monoclonal antibody 2A10 to the recombinant protein R32LR, which contains the amino acid sequence [(NANP)15NVDP]2. Immunization of mice and rabbits using the peptide (TNRNPQ)4 induced a humoral response that recognized R32LR by an enzyme-linked immunosorbent assay and P. falciparum sporozoites by an immunofluorescence assay. These results suggest that phage epitope libraries can be exploited to screen for mimotopes in the design of subunit vaccines against infectious agents.

Characterization of a sporozoite antigen common to Plasmodium falciparum and Plasmodium berghei

Previous studies demonstrated that immunization with Plasmodium falciparum sporozoites protected mice against Plasmodium berghei sporozoite infection and that this cross-protection was mediated, at least in part, by anti-sporozoite antibody. The experiments presented in this report show that serum and monoclonal antibodies derived from these protected mice identify a novel 42 54 - kDa antigen (designated Circumsporozoite Protein 2 or CSP-2) in both P. falciparum and P. berghei sporozoites. Anti-CSP-2 monoclonal antibody blocks invasion of P. falciparum and P. berghei sporozoites into hepatoma cells in vitro and binds the cell surface of sporozoites. Passive transfer of anti-CSP-2 monoclonal antibody protected mice from P. berghei sporozoite infection. Therefore, CSP-2 appears to play a role in the cross-protective immune response observed.

Control of endophagic Anopheles mosquitoes and human malaria in Guinea Bissau, West Africa by permethrin-treated bed nets

We compared the anti-mosquito and antimalarial potentialities of placebo-treated versus permethrin-impregnated bed nets in north-western Guinea Bissau. Baseline, pre-intervention entomological and parasitological data were collected during the rainy season of 1990 and bed nets were distributed shortly before the rainy season of 1991. Pairs of 3 ethnically different villages were investigated. The villages in each pair were at least 2 km apart but belonged to the same ethnic group in an ecologically similar area. After one year permethrin-treated bed nets were provided to all people in one village of each pair and placebo-treated bed nets to the other villages. About 98% of mosquitoes caught in bedrooms belonged to Anopheles gambiae and A. melas, which we consider to be the main malaria vectors in the study villages. Mean Plasmodium falciparum sporozoite rate in A. gambiae (9.6%) and A. melas (12.4%) was highest during October–November. The Plasmodium index in children 2–9 years old in the 6 villages, at the end of the rainy season 1990, ranged between 44% and 79%. Of these, 98% were identified as P. falciparum, 1% as P. malariae and 1% as mixed infections of these species. Significant reductions of Anopheles indoor resting densities and malaria parasite rates in humans were recorded in villages which had received permethrin-treated nets, but not in the control villages. The mean number of P. falciparum-infective mosquito bites received indoors in untreated villages during the rainy season was estimated to be about 4 per child and 20 per adult. This inoculation rate was reduced by at least 78% by the use of permethrin-impregnated bed nets. The malaria parasite rates and proportions of people experiencing ‘disease with fever’ decreased significantly in villages provided with permethrin-treated nets but not in the control villages. Impregnated nets may be an important tool to reduce disease and death due to malaria in Guinea Bissau.

Conservation of the Plasmodium falciparum sporozoite surface protein gene, STARP, in field isolates and distinct species of Plasmodium

The extent of structural conservation of the Plasmodium falciparum sporozoite surface protein gene, STARP, recently characterized in the T9/96 clone, has been analyzed using the polymerase chain reaction. Results from Ivory Coast and Thai clones, field isolates originating from Brazil and Kenya and laboratory-maintained strains strongly suggest that this gene has a highly conserved structure throughout this species. This structure includes a complex repetitive central domain consisting of a mosaic region followed by tandem 45-amino acid-encoding (Rp45) and 10-amino acid-encoding (Rp10) repeat regions. Limited size variation in this domain appeared to result from highly localized duplication events in the Rp45 and Rp10 regions. No size variation was observed in the 5′ and 3′ coding non-repetitive regions, but minor size polymorphism was found in the single intron at the 5′ end of the gene. No evidence was found of distinct families of polymorphic types, as has been observed with the blood-stage MSA-1, MSA-2 and S-antigens. The sequence of the STARP homologue in the phylogenetically close chimpanzee parasite, Plasmodium reichenowi, has also been elucidated and reveals high sequence conservation, although interesting differences were detected in the composition of the Rp10 region, known in P. falciparum to contain B- and T-cell epitopes. Finally, DNA hybridization reveals the presence in rodent malaria species of sequences containing homology to the STARP non-repetitive (though not the repetitive) regions, which would suggest that a similar, conserved gene may exist in these species.

Induction of murine cytotoxic T lymphocytes against Plasmodium falciparum sporozoite surface protein 2.

Sporozoite surface protein 2 has been identified as a target of malaria vaccines designed to produce protective CD8+ cytotoxic T lymphocytes (CTL) because mice immunized with mastocytoma cells expressing a fragment of Plasmodium yoelii sporozoite surface protein 2 (PySSP2) are protected against malaria by an immune response that requires CD8+ CTL. To define CTL epitopes in the Plasmodium falciparum sporozoite surface protein 2 (PfSSP2), spleen cells (SC) from mice immunized with irradiated sporozoites (irr spz) were stimulated with synthetic peptides, and these effectors were tested for cytolytic activity against peptide-pulsed, major histocompatibility complex (MHC)-matched targets. Two peptides containing CTL epitopes, A6 (Pf SSP2 3D7 214-233) and BH1 (Pf SSP2 3D7 3-11) were identified in bulk cultures of SC from immune C57BL/6 mice, and by production of CTL lines. Immunization with recombinant vaccinia expressing the full length PfSSP2 induced antigen specific, MHC-restricted, CD8+ T cell-dependent cytolytic activity against these two peptides. Finally, CTL were induced by immunization with a bacteria-derived recombinant fragment of PfSSP2 (rPfSSP2) mixed with a liposomal formulation containing a cationic lipid (Lipofectin Reagent, LPF). Induced CTL lysed target cells pulsed with peptide A6 or with LPF/rPfSSP2, but not targets pulsed with only rPfSSP2. These studies demonstrate that CTL specific to PfSSP2 are present in C57BL/6 mice and that immunization with purified rPfSSP2 delivered with LPF induces a cytotoxic T cell response.

Cloning and characterization of a novel Plasmodium falciparum sporozoite surface antigen, STARP

A novel Plasmodium falciparum sporozoite antigen, STARP (Sporozoite Threonine and Asparagine-Rich Protein), detected consistently on the surface of sporozoites obtained from laboratory strains and field isolates, has been identified and cloned, following a systematic approach aimed at isolating novel non-CS sporozoite surface antigens. The 2.0-kb STARP gene has a 5′ miniexon/large central exon structure and contains a complex repetitive region encoding multiple dispersed motifs and tandem 45- and 10-amino acid repeats. In sporozoites, transcription of the STARP gene has been conclusively demonstrated by reverse PCR and Northern blot hybridisation and the 78-kDa protein has been localized by immunofluorescence and immunoelectron microscopy to the sporozoite surface. STARP is also expressed in liver stages, as revealed by immunofluorescence assays using antisera raised either to the central repetitive region or the C-terminal non-repetitive region. Expression is also detected in early ring stages, though not in mature erythrocytic or sexual stages. Identification and elucidation of this novel antigen is a step forward in current efforts aimed at developing an effective preerythrocytic-stage malaria vaccine.

Long-Term Persistence of Sterile Immunity in a Volunteer Immunized with X-Irradiated Plasmodium falciparum Sporozoites

Three volunteers were immunized by repeated exposure to the bites of Plasmodium falciparum-infected, X-irradiated mosquitoes to characterize immunologic responses and duration of protective immunity. A primary series of immunizations had been shown previously to induce sterile immunity in these volunteers against sporozoite-induced P. falciparum malaria. In the current study, antibodies to sporozoites circulated at high levels for at least 9-12 months after the volunteers were administered booster bites from X-irradiated infective mosquitoes. One volunteer challenged a second time with P. falciparum 9 months after his last immunization was again shown to be protected, whereas all 5 control subjects developed patent infections. These results set a new standard for persistence of sterile immunity against experimental P. falciparum infection.