A phase IIa, randomized, double-blind, safety, immunogenicity and efficacy trial of Plasmodium falciparum vaccine antigens merozoite surface protein 1 and RTS,S formulated with AS02 adjuvant in healthy, malaria-naïve adults.

BACKGROUND: To improve the efficacy of Plasmodium falciparum malaria vaccine RTS,S/AS02, we conducted a study in 2001 in healthy, malaria-naïve adults administered RTS,S/AS02 in combination with FMP1, a recombinant merozoite surface-protein-1, C-terminal 42kD fragment. METHODS: A double-blind Phase I/IIa study randomized N = 60 subjects 1:1:1:1 to one of four groups, N = 15/group, to evaluate safety, immunogenicity, and efficacy of intra-deltoid half-doses of RTS,S/AS02 and FMP1/AS02 administered in the contralateral (RTS,S + FMP1-separate) or same (RTS,S + FMP1-same) sites, or FMP1/AS02 alone (FMP1-alone), or RTS,S/AS02 alone (RTS,S-alone) on a 0-, 1-, 3-month schedule. Subjects receiving three doses of vaccine and non-immunized controls (N = 11) were infected with homologous P. falciparum 3D7 sporozoites by Controlled Human Malaria Infection (CHMI). RESULTS: Subjects in all vaccination groups experienced mostly mild or moderate local and general adverse events that resolved within eight days. Anti-circumsporozoite antibody levels were lower when FMP1 and RTS,S were co-administered at the same site (35.0 µg/mL: 95 % CI 20.3-63), versus separate arms (57.4 µg/mL: 95 % CI 32.3-102) or RTS,S alone (62.0 µg/mL: 95 % CI: 37.8-101.8). RTS,S-specific lymphoproliferative responses and ex vivo ELISpot CSP-specific interferon-gamma (IFN-γ) responses were indistinguishable among groups receiving RTS,S/AS02. There was no difference in antibody to FMP1 among groups receiving FMP1/AS02. After CHMI, groups immunized with a RTS,S-containing regimen had âˆ¼ 30 % sterile protection against parasitemia, and equivalent delays in time-to-parasitemia. The FMP1/AS02 alone group showed no sterile immunity or delay in parasitemia. CONCLUSION: Co-administration of RTS,S and FMP1/AS02 reduced anti-RTS,S antibody, but did not affect tolerability, cellular immunity, or efficacy in a stringent CHMI model. Absence of efficacy or delay of patency in the sporozoite challenge model in the FMP1/AS02 group did not rule out efficacy of FMP1/AS02 in an endemic population. However, a Phase IIb trial of FMP1/AS02 in children in malaria-endemic Kenya did not demonstrate efficacy against natural infection. CLINICALTRIALS: gov identifier: NCT01556945.

RTS,S malaria vaccine efficacy and immunogenicity during Plasmodium falciparum challenge is associated with HLA genotype.

Although RTS,S remains the most advanced malaria vaccine, the factors influencing differences in vaccine immunogenicity or efficacy between individuals or populations are still poorly characterised. The analyses of genetic determinants of immunogenicity have previously been restricted by relatively small sample sizes from individual trials. Here we combine data from six Phase II RTS,S trials and evaluate the relationship between HLA allele groups and RTS,S-mediated protection in controlled human malaria infections (CHMI), using multivariate logistic or linear regression. We observed significant associations between three allele groups (HLA-A∗01, HLA-B∗08, and HLA-DRB1∗15/∗16) and protection, while another three allele groups (HLA-A∗03, HLA-B∗53, and HLA-DRB1∗07) were significantly associated with lack of protection. It is noteworthy that these 'protective' allele groups are thought to be at a lower prevalence in sub-Saharan African populations than in the UK or USA where these Phase II trials occurred. Taken together, the analyses presented here give an indication that HLA genotype may influence RTS,S-mediated protective efficacy against malaria infection.

Genetic Diversity and Protective Efficacy of the RTS,S/AS01 Malaria Vaccine.

BACKGROUND: The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus. METHODS: We used polymerase chain reaction-based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination. RESULTS: In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P=0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P=0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy. CONCLUSIONS: These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.).

Live attenuated malaria vaccine designed to protect through hepatic CD8⁺ T cell immunity.

Our goal is to develop a vaccine that sustainably prevents Plasmodium falciparum (Pf) malaria in ≥80% of recipients. Pf sporozoites (PfSPZ) administered by mosquito bites are the only immunogens shown to induce such protection in humans. Such protection is thought to be mediated by CD8(+) T cells in the liver that secrete interferon-γ (IFN-γ). We report that purified irradiated PfSPZ administered to 80 volunteers by needle inoculation in the skin was safe, but suboptimally immunogenic and protective. Animal studies demonstrated that intravenous immunization was critical for inducing a high frequency of PfSPZ-specific CD8(+), IFN-γ-producing T cells in the liver (nonhuman primates, mice) and conferring protection (mice). Our results suggest that intravenous administration of this vaccine will lead to the prevention of infection with Pf malaria.

Report of a consultation on the optimization of clinical challenge trials for evaluation of candidate blood stage malaria vaccines, 18-19 March 2009, Bethesda, MD, USA.

Development and optimization of first generation malaria vaccine candidates has been facilitated by the existence of a well-established Plasmodium falciparum clinical challenge model in which infectious sporozoites are administered to human subjects via mosquito bite. While ideal for testing pre-erythrocytic stage vaccines, some researchers believe that the sporozoite challenge model is less appropriate for testing blood stage vaccines. Here we report a consultation, co-sponsored by PATH MVI, USAID, EMVI and WHO, where scientists from all institutions globally that have conducted such clinical challenges in recent years and representatives from regulatory agencies and funding agencies met to discuss clinical malaria challenge models. Participants discussed strengthening and harmonizing the sporozoite challenge model and considered the pros and cons of further developing a blood stage challenge possibly better suited for evaluating the efficacy of blood stage vaccines. This report summarizes major findings and recommendations, including an update on the Plasmodium vivax clinical challenge model, the prospects for performing experimental challenge trials in malaria endemic countries and an update on clinical safety data. While the focus of the meeting was on the optimization of clinical challenge models for evaluation of blood stage candidate malaria vaccines, many of the considerations are relevant for the application of challenge trials to other purposes.

Plasmodium falciparum cytoadherence to human placenta: evaluation of hyaluronic acid and chondroitin 4-sulfate for binding of infected erythrocytes.

Chondroitin 4-sulfate (C4S) is known to mediate the adherence of Plasmodium falciparum infected red blood cells (IRBCs) to human placenta. Recently, hyaluronic acid (HA) has also been reported to bind IRBCs, and HA has been suggested as an additional receptor for the sequestration of IRBCs in the placenta. In this study, we assessed the adherence of 3D7 parasite strain, which has been reported to bind both C4S and HA, using highly purified clinical grade rooster comb HA, Streptococcus HA, several preparations of human umbilical cord HA (hucHA), and bovine vitreous humor HA (bvhHA). While all hucHA preparations and bvhHA bound with moderate to high density to IRBCs, the rooster comb and bacterial HAs did not bind IRBCs. IRBCs binding to the hucHA and bvhHA could be abolished by pretreatment with testicular hyaluronidase but not with Streptomyces hyalurolyticus hyaluronidase, suggesting that IRBC binding to hucHA and bvhHA was due to chondroitin sulfate (CS) contaminants in HAs. Compositional analysis confirmed the presence of CS in both hucHA and bvhHA. The CSs present in these commercial hucHA and bvhHA samples were isolated, characterized, and studied for their ability to bind IRBCs. The data suggested that IRBC adherence to hucHA and bvhHA was mediated by the CS present in these samples. However, our data did not exclude the possibility of a minor population of distinct parasite subtype adhering to HA and further studies using pure HA conjugated to proteins or lipids and placental parasite isolates should clarify whether HA is an in vivo receptor for IRBC adherence.

Gravidity-dependent production of antibodies that inhibit binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate proteoglycan during pregnancy.

During pregnancy, Plasmodium falciparum-infected erythrocytes sequester in the placenta by adhering to chondroitin 4-sulfate, creating a risk factor for both the mother and the fetus. The primigravidae are at higher risk for placental malaria than the multigravidae. This difference in susceptibility has been attributed to the lack of antibodies that block the adhesion of infected erythrocytes to placental chondroitin 4-sulfate in primigravid women. However, recent results show that many primigravidae at term have antibody levels similar to those of multigravidae, and thus the significance of antiadhesion antibodies in providing protection against malaria during pregnancy remains unclear. In this study, we analyzed plasma samples from women of various gravidities at different gestational stages for antiadhesion antibodies. The majority of women, regardless of gravidity, had similar levels of antibodies at term. Most primigravidae had low levels of or no antiadhesion antibodies prior to ~20 weeks of pregnancy and then produced antibodies. Multigravidae also lacked antibodies until ~12 weeks of pregnancy, but thereafter they efficiently produced antibodies. In pregnant women who had placental infection at term, higher levels of antiadhesion antibodies correlated with lower levels of placental parasitemia. The difference in kinetics of antibody production between primigravidae and multigravidae correlated with the prevalence of malaria in these groups, suggesting that antibodies are produced during pregnancy in response to placental infection. The early onset of efficient antibody response in multigravidae and the delayed production to antibodies in primigravidae appear to account for the gravidity-dependent differential susceptibilities of pregnant women to placental malaria.

Purification, characterization, and immunogenicity of a disulfide cross-linked Plasmodium vivax vaccine candidate antigen, merozoite surface protein 1, expressed in Escherichia coli.

The Plasmodium vivax merozoite surface protein 1 (MSP-1) 42-kDa fragment (PvMSP-1 p42) is a promising vaccine candidate antigen against the blood stage of the malarial parasite. We have developed a process for the production of this vaccine target, keeping in mind its use in human volunteers. A novel strain, Origami(DE3), of Escherichia coli with mutations in the glutathione and thioredoxin reductase genes yielded 60% more soluble PvMSP-1 p42 than the conventional E. coli BL21(DE3) strain. Recombinant PvMSP-1 p42 was purified to > or = 99% purity with a rapid two-step protocol designed for easy scaling up. The final product had a low endotoxin content and was stable in its lyophilized form. PvMSP-1 p42 was found to have the predicted primary and tertiary structures and consisted of a single conformer containing one free cysteine, as predicted. The product was recognized by conformational monoclonal antibodies against P. vivax MSP-1. Immunogenicity studies of PvMSP-1 p42 were carried out with two strains of mice and the adjuvants Montanide ISA51 and Montanide ISA720. Both formulations were found to induce high levels of immunoglobulin G1 (IgG1), IgG2b, and IgG2a antibodies along with low levels of IgG3. Lymphocytes from animals in all the PvMSP-1 p42-immunized groups showed proliferative responses upon stimulation with PvMSP-1 p42; the cytokines interleukin 2 (IL-2), gamma interferon, IL-4, and IL-10 were detected in the culture supernatants. These results indicate that PvMSP-1 p42 in combination with both of the adjuvants elicited cellular and humoral responses in mice.

Malaria rapid diagnostic devices: performance characteristics of the ParaSight F device determined in a multisite field study.

Microscopic detection of parasites has been the reference standard for malaria diagnosis for decades. However, difficulty in maintaining required technical skills and infrastructure has spurred the development of several nonmicroscopic malaria rapid diagnostic devices based on the detection of malaria parasite antigen in whole blood. The ParaSight F test is one such device. It detects the presence of Plasmodium falciparum-specific histidine-rich protein 2 by using an antigen-capture immunochromatographic strip format. The present study was conducted at outpatient malaria clinics in Iquitos, Peru, and Maesod, Thailand. Duplicate, blinded, expert microscopy was employed as the reference standard for evaluating device performance. Of 2,988 eligible patients, microscopy showed that 547 (18%) had P. falciparum, 658 (22%) had P. vivax, 2 (0.07%) had P. malariae, and 1,750 (59%) were negative for Plasmodium. Mixed infections (P. falciparum and P. vivax) were identified in 31 patients (1%). The overall sensitivity of ParaSight F for P. falciparum was 95%. When stratified by magnitude of parasitemia (no. of asexual parasites per microliter of whole blood), sensitivities were 83% (>0 to 500 parasites/microl), 87% (501 to 1,000/microl), 98% (1,001 to 5,000/microl), and 98% (>5,000/microl). Device specificity was 86%.

Sialic acid-dependent binding of baculovirus-expressed recombinant antigens from Plasmodium falciparum EBA-175 to Glycophorin A.

The Plasmodium falciparum Erythrocyte Binding Antigen-175, EBA-175, is a soluble merozoite stage parasite protein which binds to glycophorin A surface receptors on human erythrocytes. We have expressed two conserved cysteine-rich regions, region II and region VI, of this protein as soluble His-tagged polypeptides in insect cell culture, and have tested their function in erythrocyte and glycophorin A binding assays. Recombinant region II polypeptides comprised of the F2 sub-domain or the entire region II (F1 and F2 sub-domains together) bound to erythrocytes and to purified glycophorin A in a manner similar to the binding of native P. falciparum EBA-175 to human red cells. Removal of sialic acid residues from the red cell surface totally abolished recombinant region II binding, while trypsin treatment of the erythrocyte surface reduced but did not eliminate recombinant region II binding. Synthetic peptides from three discontinuous regions of the F2 sub-domain of region II inhibited human erythrocyte cell binding and glycophorin A receptor recognition. Immune sera raised against EBA-175 recombinant proteins recognized native P. falciparum-derived EBA-175, and sera from malaria-immune adults recognized recombinant antigens attesting to both the antigenicity and immunogenicity of proteins. These results suggest that the functionally-active recombinant region II domain of EBA-175 may be an attractive candidate for inclusion in multi-component asexual blood stage vaccines.

Efficacy of recombinant circumsporozoite protein vaccine regimens against experimental Plasmodium falciparum malaria.

After initial successful evaluation of the circumsporozoite-based vaccine RTS,S/SBAS2, developed by SmithKline Beecham Biologicals with the Walter Reed Army Institute of Research, protective efficacy of several regimens against Plasmodium falciparum challenge was determined. A controlled phase 1/2a study evaluated 1 or 2 standard doses of RTS,S/SBAS2 in 2 groups whose members received open-label therapy and 3 immunizations in blinded groups who received standard, one-half, or one-fifth doses. RTS,S/SBAS2 was safe and immunogenic in all groups. Of the 41 vaccinees and 23 control subjects who underwent sporozoite challenge, malaria developed in 7 of 10 who received 1 dose, in 7 of 14 who received 2 doses, in 3 of 6 who received 3 standard doses, in 3 of 7 who received 3 one-half doses, in 3 of 4 who received 3 one-fifth doses, and in 22 of 23 control subjects. Overall protective efficacy of RTS,S/SBAS2 was 41% (95% confidence interval, 22%-56%; P=.0006). This and previous studies have shown that 2 or 3 doses of RTS,S/SBAS2 protect against challenge with P. falciparum sporozoites.

Glycosylphosphatidylinositol anchors of Plasmodium falciparum: molecular characterization and naturally elicited antibody response that may provide immunity to malaria pathogenesis.

Induction of proinflammatory cytokine responses by glycosylphosphatidylinositols (GPIs) of intraerythrocytic Plasmodium falciparum is believed to contribute to malaria pathogenesis. In this study, we purified the GPIs of P. falciparum to homogeneity and determined their structures by biochemical degradations and mass spectrometry. The parasite GPIs differ from those of the host in that they contain palmitic (major) and myristic (minor) acids at C-2 of inositol, predominantly C18:0 and C18:1 at sn-1 and sn-2, respectively, and do not contain additional phosphoethanolamine substitution in their core glycan structures. The purified parasite GPIs can induce tumor necrosis factor alpha release from macrophages. We also report a new finding that adults who have resistance to clinical malaria contain high levels of persistent anti-GPI antibodies, whereas susceptible children lack or have low levels of short-lived antibody response. Individuals who were not exposed to the malaria parasite completely lack anti-GPI antibodies. Absence of a persistent anti-GPI antibody response correlated with malaria-specific anemia and fever, suggesting that anti-GPI antibodies provide protection against clinical malaria. The antibodies are mainly directed against the acylated phosphoinositol portion of GPIs. These results are likely to be valuable in studies aimed at the evaluation of chemically defined structures for toxicity versus immunogenicity with implications for the development of GPI-based therapies or vaccines.

Characterization of proteoglycans of human placenta and identification of unique chondroitin sulfate proteoglycans of the intervillous spaces that mediate the adherence of Plasmodium falciparum-infected erythrocytes to the placenta.

In pregnant women infected with Plasmodium falciparum, the infected red blood cells (IRBCs) selectively accumulate in the intervillous spaces of placenta, leading to poor fetal outcome and severe health complications in the mother. Although chondroitin 4-sulfate is known to mediate IRBC adherence to placenta, the natural receptor has not been identified. In the present study, the chondroitin sulfate proteoglycans (CSPGs) of human placenta were purified and structurally characterized, and adherence of IRBCs to these CSPGs investigated. The data indicate that the placenta contains three distinct types of CSPGs: significant quantities of uniquely low sulfated, extracellular CSPGs localized in the intervillous spaces, minor amounts of two cell-associated CSPGs, and major amounts of dermatan sulfate-like CSPGs of the fibrous tissue. Of the various CSPGs isolated from the placenta, the low sulfated CSPGs of the intervillous spaces most efficiently bind IRBCs. Based on IRBC adherence capacities and localization patterns of various CSPGs, we conclude that the CSPGs of the intervillous spaces are the receptors for placental IRBC adherence. The identification and characterization of these CSPGs provide a valuable tool for understanding the precise molecular interactions involved in placental IRBC adherence and for the development of therapeutic strategies for maternal malaria. In the accompanying paper (Alkhalil, A., Achur, R. N., Valiyaveettil, M., Ockenhouse, C. F., and Gowda, D. C. (2000) J. Biol. Chem. 275, 40357-40364), we report the structural requirements for the IRBC adherence.

Structural requirements for the adherence of Plasmodium falciparum-infected erythrocytes to chondroitin sulfate proteoglycans of human placenta.

Plasmodium falciparum infection during pregnancy results in the accumulation of infected red blood cells (IRBCs) in the placenta, leading to poor pregnancy outcome. In the preceding paper (Achur, R. N., Valiyaveettil, M., Alkhalil, A., Ockenhouse, C. F., and Gowda, D. C. (2000) J. Biol. Chem. 275, 40344-40356), we reported that unusually low sulfated chondroitin sulfate proteoglycans (CSPGs) in the intervillous spaces of the placenta mediate the IRBC adherence. In this study, we report the structural requirements for the adherence and the minimum chondroitin 4-sulfate (C4S) structural motif that supports IRBC adherence. Partially sulfated C4Ss with varying sulfate contents were prepared by solvolytic desulfation of a fully sulfated C4S. These and other nonmodified C4Ss, with different proportions of 4-, 6-, and nonsulfated disaccharide repeats, were analyzed for inhibition of IRBC adherence to the placental CSPG. C4Ss containing 30-50% 4-sulfated and 50-70% nonsulfated disaccharide repeats efficiently inhibited IRBC adherence; C6S had no inhibitory activity. Oligosaccharides of varying sizes were prepared by the partial depolymerization of C4Ss containing varying levels of 4-sulfation, and their ability to inhibit the IRBC adherence was studied. Oligosaccharides with six or more disaccharide repeats inhibited IRBC adherence to the same level as that of the intact C4Ss, indicating that a dodecasaccharide is the minimum structural motif required for optimal IRBC adherence. Of the C4S dodecasaccharides, only those with two or three sulfate groups per molecule showed maximum IRBC inhibition. These data define the structural requirements for the IRBC adherence to placental CSPGs with implications for the development of therapeutics for maternal malaria.

Adherence of Plasmodium falciparum-infected erythrocytes to chondroitin 4-sulfate.

Adherence of Plasmodium falciparum-infected erythrocytes (PRBCs) to the microvascular endothelium of specific organs and consequent sequestration is believed to be responsible for the development of malaria pathology. A number of studies have shown that cell adhesion molecules expressed on the surface of endothelial cells mediate the adherence. Recent studies indicate that a subpopulation of PRBCs adhere to chondroitin 4-sulfate (C4S). This adhesion can be effectively inhibited by C4S oligosaccharides. In pregnant women, the placenta specifically selects C4S-adherent PRBCs, and thus these phenotypes multiply and sequester in the intervillous spaces. Over successive pregnancies, women develop a protective humoral response to the C4S-adhesion phenotype. Disruption of C4S-mediated PRBCs adhesion using either a C4S oligosaccharide mimetic or an antiC4S-adhesion vaccine can be an efficient strategy for the treatment of malaria caused by C4S-adherent P. falciparum.

Long-term efficacy and immune responses following immunization with the RTS,S malaria vaccine.

The malaria sporozoite vaccine candidate RTS,S, formulated with an oil-in-water emulsion plus the immunostimulants monophosphoryl lipid A and the saponin derivative QS21 (vaccine 3), recently showed superior efficacy over two other experimental formulations. Immunized volunteers were followed to determine the duration of protective immune responses. Antibody levels decreased to between one-third and one-half of peak values 6 months after the last dose of vaccine. T cell proliferation and interferon-gamma production in vitro were observed in response to RTS,S or hepatitis B surface antigen. Seven previously protected volunteers received sporozoite challenge, and 2 remained protected (1/1 for vaccine 1, 0/1 for vaccine 2, and 1/5 for vaccine 3). The prepatent period was 10.8 days for the control group and 13.2 days for the vaccinees (P < .01). Immune responses did not correlate with protection. Further optimization in vaccine composition and/or immunization schedule will be required to induce longer-lasting protective immunity.

Phase I/IIa safety, immunogenicity, and efficacy trial of NYVAC-Pf7, a pox-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria.

Candidate malaria vaccines have failed to elicit consistently protective immune responses against challenge with Plasmodium falciparum. NYVAC-Pf7, a highly attenuated vaccinia virus with 7 P. falciparum genes inserted into its genome, was tested in a phase I/IIa safety, immunogenicity, and efficacy vaccine trial in human volunteers. Malaria genes inserted into the NYVAC genome encoded proteins from all stages of the parasite's life cycle. Volunteers received three immunizations of two different dosages of NYVAC-Pf7. The vaccine was safe and well tolerated but variably immunogenic. While antibody responses were generally poor, cellular immune responses were detected in >90% of the volunteers. Of the 35 volunteers challenged with the bite of 5 P. falciparum-infected Anopheles mosquitoes, 1 was completely protected, and there was a significant delay in time to parasite patency in the groups of volunteers who received either the low or high dose of vaccine compared with control volunteers.

Baculovirus-mediated expression of Plasmodium falciparum erythrocyte binding antigen 175 polypeptides and their recognition by human antibodies.

The erythrocyte binding antigen EBA-175 is a 175-kDa Plasmodium falciparum protein which mediates merozoite invasion of erythrocytes in a sialic acid-dependent manner. The purpose of this study was to produce recombinant EBA-175 polypeptide domains which have previously been identified as being involved in the interaction of EBA-175 with erythrocytes and to determine whether these polypeptides are recognized by malaria-specific antibodies. The eba-175 gene was cloned by PCR from genomic DNA isolated from the 3D7 strain of P. falciparum. The predicted protein sequence was highly conserved with that predicted from the published eba-175 gene sequences from the Camp and FCR-3 strains of P. falciparum and contained the F segment divergent region. Purified recombinant EBA-175 polypeptide fragments, expressed as glutathione S-transferase fusion proteins in insect cells by using the baculovirus system, were recognized by antibodies present in serum from a drug-cured, malaria-immune Aotus nancymai monkey. The fusion proteins were also recognized by antibodies present in sera from individuals residing in areas where malaria is endemic. In both cases the antibodies specifically recognized the EBA-175 polypeptide portion of the fusion proteins. Antibodies raised in rabbits immunized with the recombinant fusion proteins recognized parasite proteins present in schizont-infected erythrocytes. Our results suggest that these regions of the EBA-175 protein are targets for the immune response against malaria and support their further study as possible vaccine components.

The carboxyl-terminal cytoplasmic domain of CD36 is required for oxidized low-density lipoprotein modulation of NF-kappaB activity by tumor necrosis factor-alpha.

The binding of oxidized low-density lipoprotein (Ox LDL) by monocyte-macrophages causes pleiotropic effects, including changes in gene expression, and is thought to represent an early event in atherogenesis. The integral membrane glycoprotein CD36 appears to play a physiological role in binding and uptake of Ox LDL by monocyte-macrophages, although the molecular events associated with CD36-Ox LDL interaction are unknown. To approach this issue, we used CD36 transfected Chinese hampster ovary (CHO) cells, exposed them to Ox LDL, and determined changes in the activity of the transcription factor NF-kappaB. We report here that Ox LDL enhanced DNA binding activity of nuclear extracts to an NF-kappaB sequence following activation of CD36-producing CHO cells with the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). This enhanced DNA binding activity was inhibited by coincubation of CD36 transfected cells with the human CD36-specific antibody OKM5. We also determined that activation of NF-kappaB DNA binding activity required an intact carboxyl-terminal cytoplasmic segment on CD36. Our results support the idea that human CD36 mediates signal transduction events in response to Ox LDL.