Circumsporozoite Research Articles

Highly focused anopheline breeding sites and malaria transmission in Dakar

BackgroundUrbanization has a great impact on the composition of the vector system and malaria transmission dynamics. In Dakar, some malaria cases are autochthonous but parasite rates and incidences of clinical malaria attacks have been recorded at low levels. Ecological heterogeneity of malaria transmission was investigated in Dakar, in order to characterize the Anopheles breeding sites in the city and to study the dynamics of larval density and adult aggressiveness in ten characteristically different urban areas.MethodsTen study areas were sampled in Dakar and Pikine. Mosquitoes were collected by human landing collection during four nights in each area (120 person-nights). The Plasmodium falciparum circumsporozoite (CSP) index was measured by ELISA and the entomological inoculation rates (EIR) were calculated. Open water collections in the study areas were monitored weekly for physico-chemical characterization and the presence of anopheline larvae. Adult mosquitoes and hatched larvae were identified morphologically and by molecular methods.ResultsIn September-October 2007, 19,451 adult mosquitoes were caught among which, 1,101 were Anopheles gambiae s.l. The Human Biting Rate ranged from 0.1 bites per person per night in Yoff Village to 43.7 in Almadies. Seven out of 1,101 An. gambiae s.l. were found to be positive for P. falciparum (CSP index = 0.64%). EIR ranged from 0 infected bites per person per year in Yoff Village to 16.8 in Almadies. The An. gambiae complex population was composed of Anopheles arabiensis (94.8%) and Anopheles melas (5.2%). None of the An. melas were infected with P. falciparum. Of the 54 water collection sites monitored, 33 (61.1%) served as anopheline breeding sites on at least one observation. No An. melas was identified among the larval samples. Some physico-chemical characteristics of water bodies were associated with the presence/absence of anopheline larvae and with larval density. A very close parallel between larval and adult densities was found in six of the ten study areas.ConclusionThe results provide evidence of malaria transmission in downtown Dakar and its surrounding suburbs. Spatial heterogeneity of human biting rates was very marked and malaria transmission was highly focal. In Dakar, mean figures for transmission would not provide a comprehensive picture of the entomological situation; risk evaluation should therefore be undertaken on a small scale.

Protective CD8+ T cells against Plasmodium liver stages: immunobiology of an ‘unnatural’ immune response

Immunization with high doses of irradiated sporozoites delivered by the bites of infected mosquitoes has been shown to induce protective responses against malaria, mediated in part by CD8(+) T cells. In contrast, natural transmission involving low exposure to live sporozoite antigen fails to elicit strong immunity. In this review, we examine how irradiated sporozoite immunization breaks the natural host-parasite interaction and induces protective CD8(+) T cells. Upon biting, the malaria-infected mosquitoes deposit parasites in the skin, many of which eventually exit to the bloodstream and infect hepatocytes. However, certain antigens, including the circumsporozoite (CS) protein, remain in the skin and are presented in the draining lymph node. These antigens prime specific CD8(+) T cells, which migrate to the liver where they eliminate parasitized hepatocytes. We discuss the relevance of the different tissue compartments involved in the induction and effector phases of this response, as well as the cellular requirements for priming and memory development of CD8(+) T cells, which include a complete dependence on dendritic cells and a near absolute need for CD4(+) T-cell help. Finally, we discuss the impact of the immunodominant CS protein on this protection and the implications of these findings for vaccine design.

Effector CD8+T Lymphocytes against Liver Stages ofPlasmodium yoeliiDo Not Require Gamma Interferon for Antiparasite Activity

The protective immune response against liver stages of the malaria parasite critically requires CD8(+) T cells. Although the nature of the effector mechanism utilized by these cells to repress parasite development remains unclear, a critical role for gamma interferon (IFN-gamma) has been widely assumed based on circumstantial evidence. However, the requirement for CD8(+) T-cell-mediated IFN-gamma production in protective immunity to this pathogen has not been directly tested. In this report, we use an adoptive transfer strategy with circumsporozoite (CS) protein-specific transgenic T cells to examine the role of CD8(+) T-cell-derived IFN-gamma production in Plasmodium yoelii-infected mice. We show that despite a marginal reduction in the expansion of naive IFN-gamma-deficient CS-specific transgenic T cells, their antiparasite activity remains intact. Further, adoptively transferred IFN-gamma-deficient CD8(+) T cells were as efficient as their wild-type counterparts in limiting parasite growth in naive mice. Taken together, these studies demonstrate that IFN-gamma secretion by CS-specific CD8(+) T cells is not essential to protect mice against live sporozoite challenge.

Delivery of siRNA mediated by histidine-containing reducible polycations

Histidine containing reducible polycations based on CH 6K 3H 6C monomers (His6 RPCs), are highly effective DNA transfection agents combining pH buffering endosomal escape mechanisms with rapid unpackaging following reduction in the cytoplasm. We examined their ability to mediate siRNA uptake into cells focusing on hepatocyte delivery. Co-delivery of EGFP siRNA with pEGFP plasmid DNA reduced reporter gene expression by 85%. However while DNA transfection efficiency increased with polymer size, with 162 k His6 RPCs proving the most effective, delivery of siRNA alone to EGFP stably expressing cells was only possible using 36–80 k polymers. Analysis of particle sizes showed that 80 k polymers formed more compact siRNA complexes than 162 k polymers. The reducible nature of the polymer was necessary for siRNA activity, since siRNA combined with non-reducible polylysine showed little activity. Incorporation of a targeting peptide from the Plasmodium falciparum circumsporozoite (CS) protein onto His6 RPCs, significantly improved transfection of plasmid DNA and siRNA activity in hepatocytes, but not in most non-liver cells tested. siRNA targeted to the hepatitis B virus surface antigen delivered by CS-His6 RPC, mediated falls in both mRNA and protein expression, suggesting that this delivery system could be developed for potential therapies for viral hepatitis.

Correlation of Memory T Cell Responses against TRAP with Protection from Clinical Malaria, and CD4+ CD25high T Cells with Susceptibility in Kenyans

BackgroundImmunity to malaria develops naturally in endemic regions, but the protective immune mechanisms are poorly understood. Many vaccination strategies aim to induce T cells against diverse pre-erythrocytic antigens, but correlates of protection in the field have been limited. The objective of this study was to investigate cell-mediated immune correlates of protection in natural malaria. Memory T cells reactive against thrombospondin-related adhesive protein (TRAP) and circumsporozoite (CS) protein, major vaccine candidate antigens, were measured, as were frequencies of CD4+ CD25high T cells, which may suppress immunity, and CD56+ NK cells and γδ T cells, which may be effectors or may modulate immunity.Methodology and Principal Findings112 healthy volunteers living in rural Kenya were entered in the study. Memory T cells reactive against TRAP and CS were measured using a cultured IFNγ ELISPOT approach, whilst CD4+ CD25high T cells, CD56+ NK cells, and γδ T cells were measured by flow cytometry. We found that T cell responses against TRAP were established early in life (<5 years) in contrast to CS, and cultured ELISPOT memory T cell responses did not correlate with ex-vivo IFNγ ELISPOT effector responses. Data was examined for associations with risk of clinical malaria for a period of 300 days. Multivariate logistic analysis incorporating age and CS response showed that cultured memory T cell responses against TRAP were associated with a significantly reduced incidence of malaria (p = 0.028). This was not seen for CS responses. Higher numbers of CD4+ CD25high T cells, potentially regulatory T cells, were associated with a significantly increased risk of clinical malaria (p = 0.039).ConclusionsThese data demonstrate a role for central memory T cells in natural malarial immunity and support current vaccination strategies aimed at inducing durable protective T cell responses against the TRAP antigen. They also suggest that CD4+ CD25high T cells may negatively affect naturally acquired malarial immunity.

Impact of Recombinant Adenovirus Serotype 35 Priming versus Boosting of aPlasmodium falciparumProtein: Characterization of T- and B-Cell Responses to Liver-Stage Antigen 1

Prime-boost vaccination regimens with heterologous antigen delivery systems have indicated that redirection of the immune response is feasible. We showed earlier that T-cell responses to circumsporozoite (CS) protein improved significantly when the protein is primed with recombinant adenovirus serotype 35 coding for CS (rAd35.CS). The current study was designed to answer the question whether such an effect can be extended to liver-stage antigens (LSA) of Plasmodium falciparum such as LSA-1. Studies with mice have demonstrated that the LSA-1 protein induces strong antibody response but a weak T-cell immunity. We first identified T-cell epitopes in LSA-1 by use of intracellular gamma interferon (IFN-gamma) staining and confirmed these epitopes by means of enzyme-linked immunospot assay and pentamer staining. We show that a single immunization with rAd35.LSA-1 induced a strong antigen-specific IFN-gamma CD8(+) T-cell response but no measurable antibody response. In contrast, vaccinations with the adjuvanted recombinant LSA-1 protein induced remarkably low cellular responses but strong antibody responses. Finally, both priming and boosting of the adjuvanted protein by rAd35 resulted in enhanced T-cell responses without impairing the level of antibody responses induced by the protein immunizations alone. Furthermore, the incorporation of rAd35 in the vaccination schedule led to a skewing of LSA-1-specific antibody responses toward a Th1-type immune response. Our results show the ability of rAd35 to induce potent T-cell immunity in combination with protein in a prime-boost schedule without impairing the B-cell response.

Plasmodium Circumsporozoite Protein Promotes the Development of the Liver Stages of the Parasite

The liver stages of malaria are clinically silent but have a central role in the Plasmodium life cycle. Liver stages of the parasite containing thousands of merozoites grow inside hepatocytes for several days without triggering an inflammatory response. We show here that Plasmodium uses a PEXEL/VTS motif to introduce the circumsporozoite (CS) protein into the hepatocyte cytoplasm and a nuclear localization signal (NLS) to enter its nucleus. CS outcompetes NFkappaB nuclear import, thus downregulating the expression of many genes controlled by NFkappaB, including those involved in inflammation. CS also influences the expression of over one thousand host genes involved in diverse metabolic processes to create a favorable niche for the parasite growth. The presence of CS in the hepatocyte enhances parasite growth of the liver stages in vitro and in vivo. These findings have far reaching implications for drug and vaccine development against the liver stages of the malaria parasite.

Vector bionomics and malaria transmission in the Upper Orinoco River, Southern Venezuela

A longitudinal epidemiological and entomological study was carried out in Ocamo, Upper Orinoco River, between January 1994 and February 1995 to understand the dynamics of malaria transmission in this area. Malaria transmission occurs throughout the year with a peak in June at the beginning of the rainy season. The Annual Parasite Index was 1,279 per 1,000 populations at risk. Plasmodium falciparum infections accounted for 64% of all infections, P. vivax for 28%, and P. malariae for 4%. Mixed P. falciparum/P. vivax infections were diagnosed in 15 people representing 4% of total cases. Children under 10 years accounted for 58% of the cases; the risk for malaria in this age group was 77% higher than for those in the greater than 50 years age group. Anopheles darlingi was the predominant anopheline species landing on humans indoors with a biting peak between midnight and dawn. A significant positive correlation was found between malaria monthly incidence and mean number of An. darlingi caught. There was not a significant relationship between mean number of An. darlingi and rainfall or between incidence and rainfall. A total of 7295 anophelines were assayed by ELISA for detection of Plasmodium circumsporozoite (CS) protein. Only An. darlingi (55) was positive for CS proteins of P. falciparum (0.42%), P. malariae (0.25%), and P. vivax-247 (0.1%). The overall estimated entomological inoculation rate was 129 positive bites/person/year. The present study was the first longitudinal entomological and epidemiological study conducted in this area and set up the basic ground for subsequent intervention with insecticide-treated nets.

Prevalence and level of antibodies to the circumsporozoite protein of human malaria parasites in five states of the Amazon region of Brazil

The aim of this study was to determine the prevalence of malaria infection and antibodies against the repetitive epitopes of the circumsporozoite (CS) proteins of Plasmodium falciparum, P. malariae, P. vivax VK210, P. vivax VK247, and P. vivax-like in individuals living in the states of Rondônia, Pará, Mato Grosso, Amazonas, and Acre. Active malaria transmission was occurring in all studied sites, except in Acre. P. falciparum was the predominant species in Pará and Rondônia and P. vivax in Mato Grosso. Infection by P. malariae was low but this Plasmodium species was detected in Rondônia (3.5%), Mato Grosso (2.5%), and Pará (0.8%). High prevalence and levels of serological reactivity against the CS repeat peptides of P. falciparum were detected in Rondônia (93%) and Pará (85%). Sera containing antibodies against the CS repeat of P. malariae occurred more frequently in Rondônia (79%), Pará (76%), and Amazonas (68%). Antibodies against the repeat epitope of the standard CS protein of P. vivax VK210, P. vivax VK247, and P. vivax-like were more frequent in Rondônia, Pará, and Mato Grosso. The high frequency of reactions to P. malariae in most of the areas suggests that the infection by this Plasmodium species has been underestimated in Brazil.

Baculovirus-based Vaccination Vectors Allow for Efficient Induction of Immune Responses Against Plasmodium falciparum Circumsporozoite Protein

Baculovirus vectors are able to transduce a large variety of mammalian cell types and express transgenes placed under the control of heterologous promoters. In this study, we evaluated the potential of baculovirus vectors for malaria vaccination. To induce efficient CD4(+) and CD8(+) T-cell responses, we produced a series of vectors that display the Plasmodium falciparum circumsporozoite (CS) protein in the virion envelope and/or allow for CS expression upon transduction of mammalian cells. We found that baculovirus vectors can transduce professional antigen-presenting cells and trigger their maturation, which is a prerequisite for efficient antigen presentation. Upon intramuscular injection into mice, the vector that both displayed and expressed CS induced higher anti-CS antibody titers (of the immunoglobulin (IgG)1 and IgG2a type) and a higher frequency of interferon-gamma-producing T cells specific to CS, than the vectors which either only displayed or only expressed CS. The baculovirus CS display/expression vector was also superior in inducing CS-specific CD4(+) and CD8(+) T-cell responses in vitro using human peripheral blood mononuclear cells from naive donors. This, together with the absence of pre-existing immunity to baculoviruses in humans, the absence of viral gene expression in mammalian cells, and the relative low immunogenicity of baculovirus virions, makes these vectors promising tools for vaccination. Furthermore, the ability to produce large amounts in serum-free medium at a low cost adds a further advantage to this vector system.

A Linear Peptide Containing Minimal T- and B-Cell Epitopes ofPlasmodium falciparumCircumsporozoite Protein Elicits Protection against Transgenic Sporozoite Challenge

An effective malaria vaccine is needed to address the public health tragedy resulting from the high levels of morbidity and mortality caused by Plasmodium parasites. The first protective immune mechanism identified in the irradiated sporozoite vaccine, the "gold standard" for malaria preerythrocytic vaccines, was sporozoite-neutralizing antibody specific for the repeat region of the surface circumsporozoite (CS) protein. Previous phase I studies demonstrated that a branched peptide containing minimal T- and B-cell epitopes of Plasmodium falciparum CS protein elicited antirepeat antibody and CD4(+)-T-cell responses comparable to those observed in volunteers immunized with irradiated P. falciparum sporozoites. The current study compares the immunogenicity of linear versus tetrabranched peptides containing the same minimal T- and B-cell epitopes, T1BT*, comprised of a CS-derived universal Th epitope (T*) synthesized in tandem with the T1 and B repeats of P. falciparum CS protein. A simple 48-mer linear synthetic peptide was found to elicit antisporozoite antibody and gamma interferon-secreting T-cell responses comparable to the more complex tetrabranched peptides in inbred strains of mice. The linear peptide was also immunogenic in outbred nonhuman primates (Aotus nancymaae), eliciting antibody titers equivalent to those induced by tetrabranched peptides. Importantly, the 48-mer linear peptide administered in adjuvants suitable for human use elicited antibody-mediated protection against challenge with rodent malaria transgenic sporozoites expressing P. falciparum CS repeats. These findings support further evaluation of linear peptides as economical, safe, and readily produced malaria vaccines for the one-third of the world's population at risk of malaria infection.

Intradermal Delivery of Adenoviral Type-35 Vectors Leads to High Efficiency Transduction of Mature, CD8+ T Cell-Stimulating Skin-Emigrated Dendritic Cells

Recombinant adenovirus (Ad) type 35 (rAd35) shows great promise as vaccine carrier with the advantage of low pre-existing immunity in human populations, in contrast to the more commonly used rAd5 vector. The rAd35 vector uses CD46 as a high-affinity receptor, which, unlike the rAd5 receptor, is expressed on human dendritic cells (DC), the most powerful APCs identified to date. In this study, we show that in contrast to rAd5, rAd35 infects migrated and mature CD83+ cutaneous DC with high efficiency (up to 80%), when delivered intradermally in an established human skin explant model. The high transduction efficiency is in line with high expression levels of CD46 detected on migratory cutaneous DC, which proved to be further increased upon intradermal administration of GM-CSF and IL-4. As compared with Ad5, these Ad35 infection characteristics translate into higher absolute numbers of skin-emigrated DC per explant that both express the transgene and are phenotypically mature. Finally, we demonstrate that upon intracutaneous delivery of a rAd35 vaccine encoding the circumsporozoite (CS) protein of Plasmodium falciparum, emigrated DC functionally express and process CS-derived epitopes and are capable of activating specific CD8+ effector T cells, as evidenced by activation of an HLA-A2-restricted CS-specific CD8+ T cell clone. Collectively, these data demonstrate the utility of rAd35 vectors for efficient in vivo human DC transduction.

Safety and immunogenicity of the malaria candidate vaccines FP9 CS and MVA CS in adult Gambian men

We assessed the safety and immunogenicity of prime-boost vectors encoding the Plasmodium falciparum circumsporozoite (CS) protein expressed either in the attenuated fowl-pox virus (FP9) or modified vaccinia virus Ankara (MVA). Thirty-two adult Gambians in groups of four to eight received one, two or three doses of FP9 CS and/or MVA CS. No serious adverse event was observed following vaccination. The most immunogenic regimen was two doses of FP9 followed by a single dose of MVA 4 weeks later (an average of 1000 IFN-γ spot forming units/million PBMCs). This level of effector T-cell responses appears higher than that seen in previously reported studies of CS-based candidate malaria vaccines.

Small-Volume Detection of Plasmodium falciparum CSP Gene Using a 50-μm-Diameter Cavity with Self-Contained Electrochemistry

An electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of Plasmodium falciparum has been developed. The target molecule was a segment of the repeat sequence of the gene coding for the circumsporozoite (CSP) protein from the AF54087 gene. This analyte offers the possibility of specifically detecting P. falciparum. The assay involves attachment of a biotinylated primary DNA probe via its 5'-amine-terminus to the streptavidin-coated surface of microwells in a 96-well plate. The primary DNA probe (1(0)P, which was of two different sequences we call 1(0)P(a) and 1(0)P(b)) was used to capture the target (T, which was of two different sequences, T1 sequence 481-590 and T2 sequence 472-590 of AF54087 gene for the CSP gene) by hybridization to a complementary sequence on the target. On 1(0)P(a), 47 bases were complementary to T1 and T2 at 543-590, while on 1(0)P(b), 35 bases were complementary to T1 and T2 at 555-590. A secondary DNA probe that contained 36 bases with alkaline phosphatase (2(0)P-AP) label on the 3' end was hybridized to a complementary base sequence on the 5' end of the target. p-Aminophenol, which is enzymatically generated by the immobilized AP from p-aminophenyl phosphate (PAPP), is detected using electrochemistry. The peak current of cyclic voltammograms from a PAPP solution incubated inside the microwells modified with the complete assembly of the assay components gives a linear relationship with the concentration of the target (2-50 ng/mL, where P1 (P1a and P1b) and P2-AP concentrations are 50 ng/mL). A detection limit of 1.4 ng/mL (or 46 pM) of the DNA target was obtained. The signals of the assays were not significantly affected when performed in the presence of human hepatocytes, pig liver, or chicken serum indicating the viability of this assay in real clinical samples.

SAFETY AND ELICITATION OF HUMORAL AND CELLULAR RESPONSES IN COLOMBIAN MALARIA-NAIVE VOLUNTEERS BY A PLASMODIUM VIVAX CIRCUMSPOROZOITE PROTEIN–DERIVED SYNTHETIC VACCINE

Substantial experimental evidence indicates that the Plasmodium circumsporozoite (CS) protein has great potential as a vaccine candidate. We tested the safety and immunogenicity of vaccines composed of P. vivax CS-derived synthetic peptides. Sixty-nine healthy, malaria-naive volunteers were randomized to receive three injections of placebo or synthetic proteins N, R, or C (10, 30, or 100 microg/dose) in a double-blinded fashion. Vaccines were well tolerated and no serious adverse events were observed. Peptides N and R elicited humoral responses at all doses; peptide C elicicted these responses only at doses of 30 and 100 microg. The N peptide at a dose of 100 microg elicited the greatest antibody response. Antibodies to the three peptides recognized P. vivax sporozoites in an immunofluorescent antibody test. Peripheral blood mononuclear cells from most immunized volunteers also produced interferon-gamma upon peptide in vitro stimulation. These vaccines appear safe, well tolerated, and immunogenic in malaria-naive volunteers. Further optimization and development of this vaccine is being attempted to conduct phase II clinical trials.

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.

Anopheles (Anopheles) neomaculipalpus: a new malaria vector in the Amazon basin?

Anopheles (Anopheles) neomaculipalpus Curry (Diptera: Culicidae) collected by human landing catches and light traps in southern Venezuela were assayed by enzyme-linked immunosorbent assay (ELISA) for detection of Plasmodium circumsporozoite (CS) protein. A total of 356 An. neomaculipalpus were collected, of which three (0.84%) were positive for P. vivax, two for the variant 247 and one for the variant 210. The overall sporozoite rate in An. neomaculipalpus was similar to that for the principal vector An. (Nyssorhynchus) darlingi Root (0.82%) and higher than in An. (Nys.) marajoara Galvão & Damasceno (0.27%). This is the first report of An. neomaculipalpus naturally infected with Plasmodium parasites in Venezuela.

Mutational analysis of the GPI-anchor addition sequence from the circumsporozoite protein of Plasmodium

The plasma membrane of Plasmodium sporozoites is uniformly covered by the glycosylphosphatidylinositol (GPI)-anchored circumsporozoite (CS) protein. Sporozoites form in the mosquito midgut through a budding process that occurs within a multinucleate oocyst underneath the basal lamina of the gut. Earlier genetic studies established that normal sporozoite development requires CS. Mutant parasites lacking CS [CS (-)] do not form sporozoites. Ultrastructural analysis of the oocysts from these parasites revealed that there is an early block in the cytokinesis that occurs within the multinucleate oocysts to generate individual sporozoites. Parasites that are hypomorphic for CS expression gave rise to sporozoites with abnormal morphology. These results proved that CS plays a direct role in the maturation of oocysts and in the normal budding of sporozoites. In this article, we examined if the membrane localization of CS via a GPI-anchor, is crucial for its function during sporozoite formation. We generated three mutants in Plasmodium berghei CS, CS-DeltaGPI, CS-TM1 and CS-TM2. In CS-DeltaGPI, we deleted the signal sequence required for the addition of a GPI-anchor to CS. The resulting protein was found only in the cytoplasm of the oocyst. In CS-TM1 and CS-TM2, the GPI-anchor addition sequence of CS was substituted by the transmembrane domain and truncated (to different degrees) cytoplasmic tail of Plasmodium thrombospondin-related anonymous protein (TRAP). The resulting CS protein was detected on the plasma membrane of the oocysts. The amount of CS in the mutants was similar to that of wild type. The sporozoite budding and development were abrogated in both CS-DeltaGPI and CS-TM mutants. The ultrastructure of the mutant oocysts was indistinguishable from that of the CS (-) parasites. Our results suggest that the GPI-anchor of the CS protein is required for sporogenesis.

980. Specific Immune Response Against the Plasmodium falciparum CS Protein Mediated by Baculovirus Vectors

Top of pageAbstract Malaria is the most common tropical disease worldwide. More than 300 million people are infected and about 2-3 million die annually. The most pathogenic form, Malaria tropica, is caused by Plasmodium falciparum. This enormous world-health problem is compounded by parasites becoming resistant to drugs which are commonly used for treatment and also the complete lack of an effective vaccine. Recombinant baculovirus vectors derived from the Autographa californica nuclear polyhedrosis virus (AcNPV) are commonly used as a tool for high-level expression of recombinant proteins of interest in insect cells. Baculovirus vectors can mediate an immune response against an antigen when it is displayed on the viral surface, or when it is expressed from the baculovirus backbone using promoters which are active in mammalian cells. Their ability to accommodate very large inserts of foreign DNA, the lack of a preexisting/neutralizing immunity to baculoviruses in humans and the easy generation of high titer virus stocks are further clear advantages of this vector system for the delivery of a vaccine. In order to use baculoviruses for an induction of a specific immune response we inserted a P. falciparum circumsporozoite (CS) protein expression cassette, driven by the strong CMV promoter, into the baculoviral genome (AcNPV-CSFVI). An expression of the CS protein in antigen presenting cells (APC) should activate CD8+ T cells via MHC I presentation. The display of a second, recombinant CS peptide in the viral envelope was achieved by fusion of the CS gene to the main baculovirus surface protein gp64 (AcNPV-CS/gp64). This envelope-modified baculovirus mimics the parasite to induce CD4+ T cells and CS specific antibodies following uptake into APC and antigen presentation by MHC II. A third vector (AcNPV-CSFVI-CS/gp64) was constructed to combine expression and presentation of CS antigens in mammalian cells. Expression of the CS protein upon infection of a mouse muscle cell line with AcNPV-CSFVI or AcNPV-CSFVI-CS/gp64 was confirmed by Western blotting with CS specific antibodies. For vaccination studies, BALB/c mice were injected intramuscularly with 5|[times]|108 pfu of AcNPV-CSFVI, AcNPV-CS/gp64, AcNPV-CSFVI-CS/gp64 or 10 ug recombinant CS protein and boosted after 3 weeks with the same dose. Data of IgG1/IgG2a serum ELISAs showed high CS specific antibody titers for mice injected with AcNPV-CS/gp64. Whereas infection with AcNPV-CSFVI could induce only low levels of antibodies, the combined expression and presentation of CS antigens (AcNPV-CSFVI-CS/gp64) could elicit an even higher IgG2a response. Frequencies of CS specific CD4+ and CD8+ T cells will be analyzed by ELIspot and data will be presented. Our current efforts are aimed towards targeting baculoviruses infection to dendritic cells by incorporation of specific receptor ligands into the viral envelope.

An Immunologically Cryptic Epitope of Plasmodium falciparum Circumsporozoite Protein Facilitates Liver Cell Recognition and Induces Protective Antibodies That Block Liver Cell Invasion

Circumsporozoite, a predominant surface protein, is involved in invasion of liver cells by Plasmodium sporozoites, which leads to malaria. We have previously reported that the amino terminus region (amino acids 27-117) of P. falciparum circumsporozoite protein plays a critical role in the invasion of liver cells by the parasite. Here we show that invasion-blocking antibodies are induced by a polypeptide encoding these 91 amino acids, only when it is presented in the absence of the rest of the protein. This suggests that when present in the whole protein, the amino terminus remains immunologically cryptic. A single reactive epitope was identified and mapped to a stretch of 21 amino acids from position 93 to 113. The epitope is configurational in nature, since its recognition was affected by deleting as little as 3 amino acids from either end of the 21-residue peptide. Lysine 104, the only known polymorphic position in the epitope, affected its recognition by the antibodies, and its conversion to leucine in the protein led to a substantial loss of binding activity of the protein to the hepatocytes. This indicated that in the protein, the epitope serves as a binding ligand and facilitates the interaction between sporozoite and hepatic cells. When considered along with the observation that in its native state this motif is immunologically unresponsive, we suggest that hiding functional moieties of the protein from the immune system is an evasion strategy to preserve liver cell binding function and may be of importance in designing anti-sporozoite vaccines.