Barking up the Right Tree: Using Tree Bark to Track Airborne Particles in School Environment and Link Science to Society.

Children's exposure to air pollution affects both their health and learning skills. Fine and ultrafine particulate matter (PM2.5, PM1), notably issued from traffic sources in urban centers, belong to the most potential harmful health hazards. However their monitoring and the society's awareness on their dangers need to be consolidated. In this study, raising teacher and pupil involvement for air quality improvement in their schools environment is reached through developing a passive monitoring technique (bio-sensors made of tree bark). The experiment was implemented in two urban elementary schools situated close to a main traffic road of the city of Toulouse (South of France). Magnetic properties, carbonaceous fraction measurements, and scanning electronic microscopy (SEM-EDX) investigations were realized both on passive bio-sensors and filters issued from active sampling. We find that traffic is the main PM1 source for both outdoors and indoors at schools. Higher levels of outdoor PM in the school's environments compared to urban background are reached especially in the cold period. The schools proximity to a main traffic source and lack of ventilation are the main causes for observed PM1 accumulation in classrooms. The co-working experiment with educational teams and pupils shows that the use of bio-sensors is a driver for children empowerment to air pollution and therefore represents a potential key tool for the teachers though limiting eco-anxiety. As PM accumulation is observed in many scholar environments across Europe, the proposed methodology is a step toward a better assessment of PM impact on pupil's health and learning skills.

Imaging murine NALT following intranasal immunization with flagellin-modified circumsporozoite protein malaria vaccines.

Intranasal (IN) immunization with a Plasmodium circumsporozoite (CS) protein conjugated to flagellin, a Toll-like receptor 5 agonist, was found to elicit antibody-mediated protective immunity in our previous murine studies. To better understand IN-elicited immune responses, we examined the nasopharynx-associated lymphoid tissue (NALT) in immunized mice and the interaction of flagellin-modified CS with murine dendritic cells (DCs) in vitro. NALT of immunized mice contained a predominance of germinal center (GC) B cells and increased numbers of CD11c+ DCs localized beneath the epithelium and within the GC T-cell area. We detected microfold cells distributed throughout the NALT epithelial cell layer and DC dendrites extending into the nasal cavity, which could potentially function in luminal CS antigen uptake. Flagellin-modified CS taken up by DCs in vitro was initially localized within intracellular vesicles followed by a cytosolic distribution. Vaccine modifications to enhance delivery to the NALT and specifically target NALT antigen-presenting cell populations will advance development of an efficacious needle-free vaccine for the 40% of the world's population at risk of malaria.

Single nucleotide polymorphisms of pattern recognition receptors and chronic periodontitis.

BACKGROUND AND OBJECTIVE: Periodontitis is a multifactorial disease influenced partly by genetics. Activation of pattern recognition receptors (PRRs) can lead to the up-regulation of inflammatory pathways, resulting in periodontal tissue destruction. Hence, functional polymorphisms located in PRRs can explain differences in host susceptibility to periodontitis. This study investigated single nucleotide polymorphisms of PRRs including toll-like receptor (TLR)2 (G2408A), TLR4 (A896G), TLR9 (T1486C), TLR9 (T1237C) and CD14 (C260T) in patients with chronic periodontitis and in periodontally healthy subjects. METHODS: One-hundred and fourteen patients with chronic periodontitis and 77 periodontally healthy subjects were genotyped using TaqMan® allelic discrimination assays. Fisher's exact test and chi-square analyses were performed to compare genotype and allele frequencies. RESULTS: The frequency of subjects with the CC genotype of CD14 (C260T) (24.6% in the chronic periodontitis group vs. 13% in the periodontally healthy group) and those expressing the T allele of CD14 (C260T) (CT and TT) (75.4% in the chronic periodontitis group vs. 87% in the periodontally healthy group) was statistically different among groups (p = 0.04). Homozygocity for the C allele of the CD14 (C260T) polymorphism (CC) was associated with a two--fold increased susceptibility to periodontitis (p = 0.04; odds ratio, 2.49; 95% confidence interval, 1.06-6.26). Individuals with the CC genotype of TLR9 (T1486C) (14.9% in the chronic periodontitis group vs. 28.6% in the periodontally healthy group) and those expressing the T allele of TLR9 (T1486C) (CT and TT) (85.1% in the chronic periodontitis group vs. 71.4% in the periodontally healthy group) were also significantly differently distributed between groups without adjustment (p = 0.03). Further analysis of nonsmokers revealed a significant difference in the distribution of genotypes between groups for TLR9 (T1486C; p = 0.017) and CD14 (C260T; p = 0.03), polymorphisms again without adjustment. CONCLUSION: The CC genotype of CD14 (C260T) is related to susceptibility to chronic periodontitis in Caucasians. In addition, differences observed in the distribution of TLR9 (T1486C) genotypes between groups warrant further investigation.

Fibrinogen-neutrophil interactions in response to fMLP and Porphyromonas gingivalis fimbrial peptides.

Porphyromonas gingivalis (P.g) is the primary bacterial agent in many forms of chronic periodontitis. Since polymorphonuclear leukocytes (PMNs) are first-line responders to P.g.- induced inflammation, and fibrinogen is important for in vivo PMN in this disease, we have studied the effect of N-formyl-methionyl-leucyl-phenylalanine (fMLP) (an inflammatory stimulus), P.g. fimbriae and fimbrial peptides (based on FimA, the main structural protein of P.g. fimbriae) on PMN-fibrinogen interactions. Freshly isolated human PMNs were allowed to react with FITC-Fibrinogen and various fimbrial peptides (denoted as FimA followed by amino acid number within whole FimA protein), and FITC-Fibrinogen binding was measured using flow cytometry. Freshly isolated neutrophils were also challenged with Fibrinogen and/or fimbrial peptides to measure IL-8 secretion using ELISA. Our studies show that fibrinogen binding to PMNs is enhanced (p < 0.01) in response to fMLP as well as fimbrial peptides (FimA 61-80) containing the motif LTTE (p < 0.01) in a dose dependent manner but not in response to peptides without that motif. We also observed that fMLP and FimA 61-80 have an additive effect on fibrinogen binding to PMNs (p < 0.05), and fMLP and FimA 171-185 significantly inhibit fMLP-induced fibrinogen binding (p < 0.01). To determine of the role of inflammatory cytokines, we examined IL-8 release from PMNs in response to combinations of P. gingivalis fimbriae, fMLP and fibrinogen. In all cases, IL-8 release increased in a dose-dependent manner (p < 0.05). fMLP-fibrinogen effect on IL-8 release from PMNs was synergistic while fimbriae-fibrinogen effect was additive. In summary, PMN priming by fimbrial peptides facilitates fibrinogen-PMN interaction and may increase inflammation.

A modified hepatitis B virus core particle containing multiple epitopes of the Plasmodium falciparum circumsporozoite protein provides a highly immunogenic malaria vaccine in preclinical analyses in rodent and primate hosts.

Despite extensive public health efforts, there are presently 200 to 400 million malaria infections and 1 to 2 million deaths each year due to the Plasmodium parasite. A prime target for malaria vaccine development is the circumsporozoite (CS) protein, which is expressed on the extracellular sporozoite and the intracellular hepatic stages of the parasite. Previous studies in rodent malaria models have shown that CS repeat B-cell epitopes expressed in a recombinant hepatitis B virus core (HBc) protein can elicit protective immunity. To design a vaccine for human use, a series of recombinant HBc proteins containing epitopes of Plasmodium falciparum CS protein were assayed for immunogenicity in mice [A. Birkett, B. Thornton, D. Milich, G. A. Oliveira, A. Siddique, R. Nussenzweig, J. M. Calvo-Calle, and E. H. Nardin, abstract from the 50th Annual Meeting of the American Society of Tropical Medicine and Hygiene 2001, Am. J. Trop. Med. Hyg. 65(Suppl. 3):258, 2001; D. R. Milich, J. Hughes, J. Jones, M. Sallberg, and T. R. Phillips, Vaccine 20:771-788, 2001]. The present paper summarizes preclinical analyses of the optimal P. falciparum HBc vaccine candidate, termed ICC-1132, which contains T- and B-cell epitopes from the repeat region and a universal T-cell epitope from the C terminus of the CS protein. The vaccine was highly immunogenic in mice and in Macaca fascicularis (cynomolgus) monkeys. When formulated in adjuvants suitable for human use, the vaccine elicited antisporozoite antibody titers that were logs higher than those obtained in previous studies. Human malaria-specific CD4(+)-T-cell clones and T cells of ICC-1132-immunized mice specifically recognized malaria T-cell epitopes contained in the vaccine. In addition to inducing strong malaria-specific immune responses in naïve hosts, ICC-1132 elicited potent anamnestic antibody responses in mice primed with P. falciparum sporozoites, suggesting potential efficacy in enhancing the sporozoite-primed immune responses of individuals living in areas where malaria is endemic.

Periodontal infections contribute to elevated systemic C-reactive protein level.

BACKGROUND: Periodontitis is a local inflammatory process mediating destruction of periodontal tissues triggered by bacterial insult. However, this disease is also characterized by systemic inflammatory host responses that may contribute, in part, to the recently reported higher risk for cardiovascular disease (CVD) among patients with periodontitis. Moderate elevation of C-reactive protein (CRP) has been found to be a predictor of increased risk for CVD. Elevated CRP levels in periodontal patients have been reported by several groups. In this study, we examined whether CRP plasma levels are increased in periodontitis and if there is a relation to severity of periodontal disease and to the periodontal microflora. METHODS: CRP serum levels were assessed using radial immunodiffusion assay in 174 subjects, 59 with moderate mean clinical attachment loss (AL) (2.39+/-0.29 mm) and 50 with high AL (3.79+/-0.86 mm) as compared to 65 periodontally healthy controls (AL, 1.74+/-0.18 mm). Clinical attachment loss, probing depths, and percentage of periodontal pocket sites > or =5 mm were measured. The presence of periodontal pathogens Porphyromonas gingivalis (P.g.), Prevotella intermedia (P.i.), Campylobacter recta (C.r.), and Bacteroides forsythus (B.f.) in subgingival plaque samples was measured by immunofluorescence microscopy. RESULTS: Statistically significant increases in CRP levels were observed in subjects with periodontal disease when compared to healthy controls (P= 0.036). Subjects with high levels of mean clinical attachment loss had significantly higher mean CRP levels (4.06+/-5.55 mg/l) than controls (1.70+/-1.91 mg/l), P= 0.011. The CRP levels were adjusted for factors known to be associated with elevated CRP, including age, smoking, body mass index (BMI), triglycerides, and cholesterol. Age and BMI were found to be significant covariates. The reported range for CRP as a risk factor for CVD, peripheral vascular diseases, or stroke is 1.34 mg/l to 6.45 mg/l and the mean of this range is 3 mg/l. The percentage of subjects with elevated levels of CRP > or = 3 mm was significantly higher in the high clinical AL group (38%; 95% Cl: 26.7%, 49.3%) when compared to the control group (16.9%; 95% CI: 9.25%, 24.5%), P= 0.011. The presence of periodontal pathogens P.g., P.i., C.r., and B.f. in subgingival samples was positively associated with elevated CRP levels (P= 0.029). CONCLUSIONS: The extent of increase in CRP levels in periodontitis patients depends on the severity of the disease after adjusting for age, smoking, body mass index, triglycerides, and cholesterol. Also, there are elevated levels of CRP associated with infection with subgingival organisms often associated with periodontal disease, including P.g., P.i., C.r., and B.f. Recent investigations emphasized the role of moderate elevated CRP plasma levels as a risk factor for CVD. The positive correlation between CRP and periodontal disease might be a possible underlying pathway in the association between periodontal disease and the observed higher risk for CVD in these patients.

Development and validation of a column-switching high-performance liquid chromatographic method for the determination of sanfetrinem in rat and dog plasma by direct injection.

A direct injection column-switching HPLC method was developed and validated for quantification of sanfetrinem in rat and dog plasma. Following dilution with buffer, samples were directly injected onto the system. The analyte was retained in an enrichment column while endogenous plasma components were eluted to waste. Sanfetrinem was then back-flushed to the analytical column for separation and quantification with an ultraviolet detector. Sample batch size was increased by adding a washing phase of the enrichment column and by alternating the injections between two enrichment columns. The method is very simple and sample preparation is minimal. The method has been fully validated and shown to be specific, accurate and reproducible.

A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types.

This open-labeled phase I study provides the first demonstration of the immunogenicity of a precisely defined synthetic polyoxime malaria vaccine in volunteers of diverse HLA types. The polyoxime, designated (T1BT(*))(4)-P3C, was constructed by chemoselective ligation, via oxime bonds, of a tetrabranched core with a peptide module containing B cell epitopes and a universal T cell epitope of the Plasmodium falciparum circumsporozoite protein. The triepitope polyoxime malaria vaccine was immunogenic in the absence of any exogenous adjuvant, using instead a core modified with the lipopeptide P3C as an endogenous adjuvant. This totally synthetic vaccine formulation can be characterized by mass spectroscopy, thus enabling the reproducible production of precisely defined vaccines for human use. The majority of the polyoxime-immunized volunteers (7/10) developed high levels of anti-repeat Abs that reacted with the native circumsporozoite on P. falciparum sporozoites. In addition, these seven volunteers all developed T cells specific for the universal epitope, termed T(*), which was originally defined using CD4(+) T cells from protected volunteers immunized with irradiated P. falciparum sporozoites. The excellent correlation of T(*)-specific cellular responses with high anti-repeat Ab titers suggests that the T(*) epitope functioned as a universal Th cell epitope, as predicted by previous peptide/HLA binding assays and by immunogenicity studies in mice of diverse H-2 haplotypes. The current phase I trial suggests that polyoximes may prove useful for the development of highly immunogenic, multicomponent synthetic vaccines for malaria, as well as for other pathogens.

The role of inflammatory and immunological mediators in periodontitis and cardiovascular disease.

Epidemiological studies have implicated periodontitis (PD) as a risk factor for development of cardiovascular disease (CVD). Persistent infections such as periodontitis induce inflammatory and immune responses which may contribute to coronary atherogenesis, and, in conjunction with other risk factors, may lead to coronary heart disease (CHD). In this review, mechanisms are described that may help explain the association between periodontal infections and CHD. Periodontal diseases are bacterial infections associated with bacteremia, inflammation, and a strong immune response, all of which may represent significant risk factors for the development of atherogenesis, CHD, and myocardial infarction (MI). Several mechanisms may participate in this association, including those induced by oral organisms, and those associated with host response factors. This review will focus on host factors. Oral pathogens and inflammatory mediators (such as interleukin [IL]-1 and tumor necrosis factor [TNF]-alpha) from periodontal lesions intermittently reach the bloodstream inducing systemic inflammatory reactants such as acute-phase proteins, and immune effectors including systemic antibodies to periodontal bacteria. This review will describe the potential role of various inflammatory as well as immunologic factors that may play a role in periodontitis as a possible risk factor for CHD.

Synthetic malaria peptide vaccine elicits high levels of antibodies in vaccinees of defined HLA genotypes.

A multiple antigen peptide (MAP) malaria vaccine containing minimal Plasmodium falciparum circumsporozoite protein repeat epitopes was assessed for safety and immunogenicity in volunteers of known class II genotypes. The MAP/alum/QS-21 vaccine formulation elicited high levels of parasite-specific antibodies in 10 of 12 volunteers expressing DQB1*0603, DRB1*0401, or DRB1*1101 class II molecules. In contrast, volunteers of other HLA genotypes were low responders or nonresponders. A second study of 7 volunteers confirmed the correlation of class II genotype and high responder phenotype. This is the first demonstration in humans that a peptide vaccine containing minimal T and B cell epitopes composed of only 5 amino acids (N, A, V, D, and P) can elicit antibody titers comparable to multiple exposures to irradiated P. falciparum-infected mosquitoes. Moreover, the high-responder phenotypes were predicted by analysis of peptide/HLA interactions in vitro, thus facilitating the rational design of epitope-based peptide vaccines for malaria, as well as for other pathogens.

Interleukin 8, neutrophil-activating peptide-2 and GRO-alpha bind to and elicit cell activation via specific and different amino acid residues of CXCR2.

The objective of this investigation was to determine the amino acid residues of the human neutrophil CXC chemokine receptor-2 (CXCR2) that are critical for binding the ligands interleukin 8 (IL-8), neutrophil-activating peptide-2 (NAP-2), and growth-related protein alpha (GROalpha) and critical for receptor-mediated signal transduction. Charged residues of the amino terminus and the first extracellular loop of CXCR2 were targeted for point mutagenesis studies. Seven separate CXCR2 mutants (Glu7, Asp9, Glu12, Asp13, Lys108, Asn110, and Lys120, all to Ala) were generated. Based on the Scatchard analysis of radioligand binding studies, the following amino acids were deemed critical for ligand binding: (i) Asp9, Glu12, Lys108, and Lys120 for IL-8 and (ii) Glu7, Asp9, and Glu12 for GROalpha. Point mutations in the amino terminus domain (Asp9 and Glu12) and the first extracellular loop (Lys108, Asn110, and Lys120) of CXCR2 reduced cell activation to all three ligands as measured by changes in intracellular calcium concentration. In conclusion, high-affinity binding of IL-8, NAP-2, and GROalpha to CXCR2 involves interaction with specific and different amino acid residues of CXCR2. Furthermore, we propose that the CXCR2 amino acid residues required for cell activation are not necessarily the same residues required for ligand binding.

Pyk2 and Syk participate in functional activation of granulocytic HL-60 cells in a different manner.

The roles of the protein tyrosine kinases Pyk2 (also called RAFTK or CAK beta) and Syk in the process of functional activation of human myeloid cells were examined. During granulocytic differentiation of HL-60 cells with dimethyl sulfoxide (DMSO), the amounts of Pyk2 and beta2 integrin increased, whereas the amount of Syk was abundant before differentiation and did not change during differentiation. When the granulocytic cells were stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), tyrosine phosphorylation of Pyk2 occurred promptly and subsequent association of Pyk2 with beta2 integrin was detected. In contrast, Syk was not tyrosine phosphorylated by fMLP stimulation but constitutively associated with beta2 integrin. Stimulation with fMLP also caused the alteration of beta2 integrin to an activated form, a finding that was confirmed by the observation of fMLP-induced cell attachment on fibrinogen-coated dishes and inhibition of this attachment by pretreatment with anti-beta2 integrin antibody. Cell attachment to fibrinogen caused the enhanced tyrosine phosphorylation of Pyk2 and the initial tyrosine phosphorylation of Syk, which was also inhibited by pretreatment with anti-beta2 integrin antibody. In vitro kinase assays revealed that Pyk2 and Syk represented kinase activities to induce tyrosine phosphorylation of several molecules in the anti-beta2 integrin immunoprecipitates of the attached cells. These results showed that Pyk2 is involved in the functional activation of granulocytic cells in 2 signaling pathways: an fMLP receptor-mediated "inside-out" signaling pathway that might cause beta2 integrin activation and a subsequent beta2 integrin-mediated "outside-in" signaling pathway. Syk was activated in relation to cell attachment to fibrinogen as a result of "outside-in" signaling, although it was already associated with beta2 integrin before fMLP stimulation. (Blood. 2000;96:1733-1739)

Chemotactic-like receptors and Abeta peptide induced responses in Alzheimer's disease.

Evidence suggests that beta-amyloid (Abeta) has chemokine-like properties and may act through formyl chemotactic receptors (FPR) to induce pathophysiologically important functional changes in Alzheimer's disease (AD) microglia. We have shown that Abeta 1-42, fibrillar Abeta 1-40, and Abeta 25-35 potentiate the release of interleukin-1beta (IL-1beta) from LPS activated human THP-1 monocytes [26] and LPS primed rat microglia. Moreover, Abeta-stimulated IL-1beta secretion seems to be receptor mediated because it is calcium dependent and requires activation of specific G-proteins [27]. Thus, we have evaluated the ability of Abeta 1-42 to mimic formyl chemotactic peptides in stimulating IL-1beta release from THP-1 monocytes. Several of the formyl chemotactic peptides and Abeta 1-42 significantly enhanced IL-1beta production in THP-1 monocytes. In contrast, a formyl chemotactic receptor antagonist inhibited Abeta 1-42-induced IL-1beta release from both human THP-1 monocytes and primary rat microglia. Further, primary rat microglia grown in culture expressed FPR as demonstrated by immunocytochemistry. Given the multiple pathophysiologic roles IL-1beta may play in AD, agents that block Abeta interactions with formyl chemotactic receptors on microglia might be important antiinflammatory therapeutic targets.

Isolation and characterization of a human neutrophil aggregation defective mutant of Fusobacterium nucleatum.

Fusobacterium nucleatum is known to adhere to human polymorphonuclear neutrophils (PMNs) and cause them to aggregate. In this study, we isolated a spontaneously occurring aggregation defective (AGG(-)) mutant and this mutant will be used for future study of the interactions between this bacterium and human PMN. Genomic DNA fingerprinting by random-primed polymerase chain reaction method revealed a difference between the parent strain and the AGG(-) mutant. This mutant also showed an altered phenotype in both microbicidal and phagocytic assays, suggesting that the bacterial factor involved in the aggregation may also be very important for the phagocytosis and, subsequently, the killing by human PMNs. Further study of this mutant may help to clarify the molecular mechanisms of the interaction between this pathogen and human PMNs.

Identification and characterization of human immunoglobulin G Fc receptors of Fusobacterium nucleatum.

Several human pathogens express components which can bind to the Fc portion of immunoglobulins. This study was undertaken to characterize the human immunoglobulin G (IgG) Fc-binding activity of Fusobacterium nucleatum, a suspected pathogen involved in periodontal diseases. Fc-binding activity was detected using whole-cell, cell envelope and outer membrane fractions, and it was found to be associated with polypeptides of 40 kDa and 42 kDa, respectively. Amino terminal sequencing of these components revealed them to be homologous to the bacterial porin encoded by fomA gene. Further sequencing of internal peptide fragments obtained by CNBr cleavage suggested that these two proteins are probably isoforms. In summary, we show that a porin-like protein on the surface of F. nucleatum can bind the Fc fragment of the human immunoglobulin G, and this protein may act as a virulence factor to facilitate this bacterium in evading host immune surveillance system.

Single nucleotide polymorphisms of the N-formyl peptide receptor in localized juvenile periodontitis.

BACKGROUND: Neutrophils from patients with localized juvenile periodontitis (LJP) exhibit decreased binding and responsiveness to various chemotactic agents, including N-formyl-1-methionyl-1-leucyl-1-phenylalanine (FMLP). This altered reaction of neutrophils is thought to account in part for the increased susceptibility of LJP patients to infections by periodontal organisms. Receptors for FMLP are involved in the activation and the subsequent response to certain chemotactic stimuli. METHODS: In order to determine if this decreased response is due to a genetic variation in the receptor, we directly compared DNA encoding the FMLP receptor from controls matched for gender and race and LJP patients by single-strand conformation polymorphism analysis (SSCP). RESULTS: Using this technique, we observed a characteristic SSCP pattern in 29 out of 30 patient samples in the FMLP receptor DNA. This pattern differed from those obtained from the 20 control subjects as well as 31 patients with adult periodontitis. DNA sequencing of 30 patients indicated single nucleotide polymorphisms (SNPs) in the FMLP receptor DNA from the LJP patients when compared to 20 controls (P = 0.0005). Two single nucleotide base alterations were consistently seen: either a thymine to cytosine substitution at base 329 in 17 LJP patients or a cytosine to guanine substitution at base 378 in 5 LJP patients. A combination of both alterations were seen in 7 LJP patients. Both alterations resulted in amino acid changes in the second intracellular loop of the receptor, specifically phenylalanine to serine at residue 110 and cysteine to tryptophan at residue 126. This region of the FMLP receptor has recently been shown to play a role in ligand binding and G-protein activation. CONCLUSIONS: This study suggests that a molecular alteration in the second intracellular loop of the FMLP receptor molecules in LP patients may play a role in the decreased chemotactic activity reported for some LJP patients.

Human formyl peptide receptor function role of conserved and nonconserved charged residues.

In this study, we investigated the role of charged residues in ligand binding interactions of f-Met-Leu-Phe receptors (FPR). Charged residues of FPR, both conserved and nonconserved, which are located close to the membrane interface were mutated to alanine to determine their role in ligand binding. The mutated residues belonged to specific domains of FPR which have previously been implicated in FPR ligand binding interactions. We demonstrate that nonconserved charged residues such as Arg84, Lys85, Arg205 and Asp284 and conserved charge residue Arg163 seem to play a role in ligand binding. However, alteration of nonconserved charged residue Asp106 did not have any effect. In conclusion, specific charged residues of FPR, both conserved nonconserved, may contribute to FPR function either directly or indirectly.

Preclinical evaluation of a synthetic Plasmodium falciparum MAP malaria vaccine in Aotus monkeys and mice.

Multiple antigen peptides (MAPs) containing epitopes of the major surface protein of the malaria sporozoite, the circumsporozoite (CS) protein, have been shown in previous studies to elicit antibody-mediated protection against sporozoite challenge in experimental murine and simian hosts. For the preparation for a phase I trial of a P. falciparum (T1B)4 MAP, which contains T and B cell epitopes from the CS repeat region, pre-clinical immunogenicity and adjuvant formulation studies were carried out in mice and Aotus monkeys. The (T1B)4 MAP was found to be immunogenic in three different species of owl monkeys, Aotus nancymae, A. vociferans and A. nigriceps. Optimal antibody responses were obtained in A. nancymae immunized s.c. with (T1B)4 MAP emulsified in Freund's, in which peak titers of over 10(6) were obtained in individual monkeys. MAP immunized A. vociferans also developed high levels of anti-sporozoite antibodies, although the kinetics and the magnitude of the response differed from A. nancymae. (T1B)4 MAP adsorbed to alum (aluminum hydroxide), a formulation that is acceptable for human use, was less immunogenic in naive A. nancymae, as well as A. nigriceps. The injection of MAPs/alum, however, significantly enhanced antibody responses in sporozoite-primed monkeys, suggesting that the administration of the MAP vaccine may be an effective means to increase the low levels of antibody present in individuals living in malaria endemic areas. The addition of a co-adjuvant QS-21, a purified saponin, significantly increased the immunogenicity of the alum-adsorbed MAP in both mice and monkeys, providing a vaccine formulation suitable for phase I trials in human volunteers.

Pre-erythrocytic malaria vaccine: mechanisms of protective immunity and human vaccine trials.

In order to provide a rational basis for the development of a pre-erythrocytic malaria vaccine we have aimed at: (a) elucidating the mechanisms of protection, and (b) identifying vaccine formulations that best elicit protection in experimental animals and humans. Based on earlier successful immunization of experimental animals with irradiated sporozoites, human volunteers were exposed to the bites of large numbers of Plasmodium falciparum or P. vivax infected irradiated mosquitoes. The result of this vaccine trial demonstrated for the first time that a pre-erythrocytic vaccine, administered to humans, can result in their complete resistance to malaria infection. However, since infected irradiated mosquitoes are unavailable for large scale vaccination, the alternative is to develop subunit vaccines. The human trials using irradiated sporozoites provided valuable information on the human immune responses to pre-erythrocytic stages and studies on mice an excellent experimental model to characterize protective immune mechanisms. The circumsporozoite protein, the first pre-erythrocytic antigen identified, is present in all malaria species, displaying a similar structure, with a central region of repeats, and two conserved regions, essential for parasite development. Most pre-erythrocytic vaccine candidates are based on the CS protein, expressed in various cell lines, microorganisms, and recently the corresponding DNA. We and others have identified CS-specific B and T cell epitopes, recognized by the rodent and human immune systems, and used them for the development of synthetic vaccines. We used synthetic peptide vaccines, multiple antigen peptides and polyoximes, for immunization, first in experimental animals, and recently in two human safety and immunogenicity trials. We also report here on our work on T cell mediated immunity, particularly the protection of mice immunized with viral vectors expressing CS-specific cytotoxic CD8+ T cell epitopes, and the striking booster effect of recombinant vaccinia virus. To what degree CD8+ T cells, and/or other T cells specific for sporozoites and/or liver stage epitopes, contribute to pre-erythrocytic protective immunity in humans, remains to be determined.

Immunogenicity of chimeric multiple antigen peptides based on Plasmodium falciparum antigens: impact of epitope orientation.

Assembly of B and T epitopes in multiple antigen peptides (MAP) can bypass genetically predisposed unresponsiveness to B epitopes. Although the underlying mechanisms are unknown, B-cell responses to such diepitope MAP are influenced by intramolecular epitope orientation. In this study, MAP constructs were synthesized, encompassing two epitopes derived from the Plasmodium falciparum antigens circumsporozoite protein (CS) and Pf332. In addition to B epitopes, the sequences comprised T epitopes restricted to mouse H-2b (CS) or to H-2d and H-2k (Pf332) haplotypes. Congenic H-2b, H-2d and H-2k Balb mice were immunized with MAP in which the two epitopes were arranged either tandemly or in parallel. Tandemly arranged (B-T)4 MAP, in which the relevant T epitope was positioned adjacent to the lysine core [(Pf332-CS)4-core for H-2b mice and (CS-Pf332)4-core for H-2d and H-2k mice], elicited the most potent antibody responses in terms of reactivity to both epitopes. Additionally, the (B-T)4 constructs were generally most efficient in recalling proliferative T-cell responses in vitro, irrespective of the MAP used for in vivo priming. As high antibody titers were generated to both epitopes, the position of B epitopes in the constructs does not appear to be critical for an efficient B-cell response. Rather, the association of strong B- and T-cell responses to the (B-T)4 MAP constructs suggests that the intramolecular position of the relevant T epitope determines the magnitude of specific antibody production.