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The N-terminal domain of the circumsporozoite protein (CSP) has been largely neglected in the search for a malaria vaccine in spite of being a target of inhibitory antibodies and protective T cell responses in mice. Thus, in order to develop this region as a vaccine candidate to be eventually associated with other candidates and, in particular, with the very advanced C-terminal counterpart, synthetic constructs representing N- and C-terminal regions of Plasmodium falciparum and Plasmodium berghei CSP were administered as single or combined formulations in mice. We show that the antisera generated against the combinations inhibit sporozoite invasion of hepatocytes in vitro better than antisera against single peptides. Furthermore, two different P. falciparum CSP N-terminal constructs (PfCS22-110 and PfCS65-110) were recognized by serum samples from people living in malaria-endemic regions. Importantly, recognition of the short N-terminal peptide (PfCS65-110) by sera from children living in a malaria-endemic region was associated with protection from disease. Taken together, these results underline the potential of using such fragments as malaria vaccine candidates.

A new method for oxidative folding of synthetic polypeptides assembled by stepwise solid phase synthesis is introduced. Folding is obtained in excellent yields by reacting S-tert-butylthiolated polypeptides with a 100-fold molar excess of cysteine at 37 degrees C in a slightly alkaline buffer containing chaotropic salts, and in the presence of air-oxygen. This novel protocol has been applied to the folding of S-tert-butylthiolated human thymus and activation-regulated chemokine (hu-TARC) derivatives as well as to larger segments of Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins. Folded P. falciparum polypeptides have been used as substrates of endoproteinase Glu-C (Glu-C) and endoproteinase Asp-N (Asp-N) in an attempt to identify their disulfide connectivities. Particular practical advantages of the present method are (i) easy purification and storage of the S-protected peptide derivatives, (ii) elimination of the risk of cysteine alkylation during the acidolytic cleavage deprotection and resin cleavage steps, (iii) possibility to precisely evaluate the extent of folding and disulfide bond formation by mass spectrometry, and (iv) facile recovery of the final folded product., (Copyright 2008 European Peptide Society and John Wiley & Sons, Ltd.)

The vaccine potential of Plasmodium falciparum liver stage antigen-3 (LSA3) was investigated in Aotus monkeys using two long synthetic peptides corresponding respectively to an N-terminal non-repeat peptide (NRP) and repeat 2 (R2) region of the LSA3, adjuvanted by ASO2. Both 100-222 (NRP) and 501-596 repeat peptides induced effector B- and T-cell responses in terms of antigen-driven antibodies and/or specific IFN-gamma secretion. Animals challenged with P. falciparum sporozoites were protected following immunization with either the NRP region alone or the NRP combined with the R2 repeat region, as compared with controls receiving the adjuvant alone. These results indicate that the NRP may be sufficient to induce full, sterile protection and confirm the vaccine potential of LSA3 previously demonstrated in chimpanzees and in Aotus.

CD8(+) T cells play a major role in the protective immune response against the liver stage of malaria. It was previously shown that the circumsporozoite protein (CSP) is processed and presented to specific T cells by both traversed and infected hepatocytes, but their respective antigen processing requirements were not completely defined. In the present study, we show that in vitro processing of the Plasmodium berghei CSP by infected mouse primary hepatocytes is exclusively dependent on proteasomes, while aspartic proteases are also needed in the case of traversed hepatocytes.

We have recently described 95 predicted alpha-helical coiled-coil peptides derived from putative Plasmodium falciparum erythrocytic stage proteins. Seventy peptides recognized with the highest level of prevalence by sera from three endemic areas were selected for further studies. In this study, we sequentially examined antibody responses to these synthetic peptides in two cohorts of children at risk of clinical malaria in Kilifi district in coastal Kenya, in order to characterize the level of peptide recognition by age, and the role of anti-peptide antibodies in protection from clinical malaria. Antibody levels from 268 children in the first cohort (Chonyi) were assayed against 70 peptides. Thirty-nine peptides were selected for further study in a second cohort (Junju). The rationale for the second cohort was to confirm those peptides identified as protective in the first cohort. The Junju cohort comprised of children aged 1-6 years old (inclusive). Children were actively followed up to identify episodes of febrile malaria in both cohorts. Of the 70 peptides examined, 32 showed significantly (p<0.05) increased antibody recognition in older children and 40 showed significantly increased antibody recognition in parasitaemic children. Ten peptides were associated with a significantly reduced odds ratio (OR) for an episode of clinical malaria in the first cohort of children and two of these peptides (LR146 and AS202.11) were associated with a significantly reduced OR in both cohorts. LR146 is derived from hypothetical protein PFB0145c in PlasmoDB. Previous work has identified this protein as a target of antibodies effective in antibody dependent cellular inhibition (ADCI). The current study substantiates further the potential of protein PFB0145c and also identifies protein PF11&#95;0424 as another likely target of protective antibodies against P. falciparum malaria.

Plasmodium sporozoites traverse several host cells before infecting hepatocytes. In the process, the plasma membranes of the cells are ruptured, resulting in the release of cytosolic factors into the microenvironment. This released endogenous material is highly stimulatory/immunogenic and can serve as a danger signal initiating distinct responses in various cells. Thus, our study aimed at characterizing the effect of cell material leakage during Plasmodium infection on cultured mouse primary hepatocytes and HepG2 cells. We observed that wounded cell-derived cytosolic factors activate NF-kappaB, a main regulator of host inflammatory responses, in cells bordering wounded cells, which are potential host cells for final parasite infection. This activation of NF-kappaB occurred shortly after infection and led to a reduction of infection load in a time-dependent manner in vitro and in vivo, an effect that could be reverted by addition of the specific NF-kappaB inhibitor BAY11-7082. Furthermore, no NF-kappaB activation was observed when Spect(-/-) parasites, which are devoid of hepatocyte traversing properties, were used. We provide further evidence that NF-kappaB activation causes the induction of inducible NO synthase expression in hepatocytes, and this is, in turn, responsible for a decrease in Plasmodium-infected hepatocytes. Furthermore, primary hepatocytes from MyD88(-/-) mice showed no NF-kappaB activation and inducible NO synthase expression upon infection, suggesting a role of the Toll/IL-1 receptor family members in sensing cytosolic factors. Indeed, lack of MyD88 significantly increased infection in vitro and in vivo. Thus, host cell wounding due to parasite migration induces inflammation which limits the extent of parasite infection.

Invariant NKT (iNKT) cells are potent activators of DCs, NK cells, and T cells, and their antitumor activity has been well demonstrated. A single injection of the high-affinity CD1d ligand alpha-galactosylceramide (alphaGalCer) leads to short-lived iNKT cell activation followed, however, by long-term anergy, limiting its therapeutic use. In contrast, we demonstrated here that when alphaGalCer was loaded on a recombinant soluble CD1d molecule (alphaGalCer/sCD1d), repeated injections led to sustained iNKT and NK cell activation associated with IFN-gamma secretion as well as DC maturation in mice. Most importantly, when alphaGalCer/sCD1d was fused to a HER2-specific scFv antibody fragment, potent inhibition of experimental lung metastasis and established s.c. tumors was obtained when systemic treatment was started 2-7 days after the injection of HER2-expressing B16 melanoma cells. In contrast, administration of free alphaGalCer at this time had no effect. The antitumor activity of the CD1d-anti-HER2 fusion protein was associated with HER2-specific tumor localization and accumulation of iNKT, NK, and T cells at the tumor site. Targeting iNKT cells to the tumor site thus may activate a combined innate and adaptive immune response that may prove to be effective in cancer immunotherapy.

Objectives: To examine whether the humoural response to malaria vaccine candidate antigens, Plasmodium falciparum [circumsporozoite repetitive sequence (NANP)(5) GLURP fragments (R0 and R2) and MSP3] varies with the level of malaria transmission and to determine whether the antibodies (IgG) present at the beginning of the malaria transmission season protect against clinical malaria., Methods: Cross-sectional surveys were conducted to measure antibody response before, at the peak and at the end of the transmission season in children aged 6 months to 10 years in two villages with different levels of malaria transmission. A cohort study was performed to estimate the incidence of clinical malaria., Results: Antibodies to these antigens showed different seasonal patterns. IgG concentrations to any of the four antigens were higher in the village with high entomological inoculation rate. Multivariate analysis of combined data from the two villages indicated that children who were classified as responders to the selected antigens were at lower risk of clinical malaria than children classified as non-responders [(NANP)(5) (incidence rate ratio (IRR) = 0.65, 95% CI: 0.46-0.92; P = 0.016), R0 (IRR = 0.69, 95% CI: 0.48-0.97; P = 0.032), R2 (IRR = 0.73, 95% CI: 0.50-1.06; P = 0.09), MSP3 (IRR = 0.52, 95% CI: 0.32-0.85; P = 0.009)]. Fitting a model with all four antibody responses showed that MSP3 looked the best malaria vaccine candidate (IRR = 0.63; 95% CI: 0.38-1.05; P = 0.08)., Conclusion: Antibody levels to the four antigens are affected by the intensity of malaria transmission and associated with protection against clinical malaria. It is worthwhile investing in the development of these antigens as potential malaria vaccine candidates.

Background: Research aimed at developing vaccines against infectious diseases generally seeks to induce robust immune responses to immunodominant antigens. This approach has led to a number of efficient bacterial and viral vaccines, but it has yet to do so for parasitic pathogens. For malaria, a disease of global importance due to infection by Plasmodium protozoa, immunization with radiation-attenuated sporozoites uniquely leads to long lasting sterile immunity against infection. The circumsporozoite protein (CSP), an important component of the sporozoite's surface, remains the leading candidate antigen for vaccines targeting the parasite's pre-erythrocytic stages. Difficulties in developing CSP-based vaccines that reproduce the levels of protection afforded by radiation-attenuated sporozoites have led us to question the role of CSP in the acquisition of sterile immunity. We have used a parasite transgenic for the CSP because it allowed us to test whether a major immunodominant Plasmodium antigen is indeed needed for the induction of sterile protective immunity against infection., Methodology/main Findings: We employed a P. berghei parasite line that expresses a heterologous CSP from P. falciparum in order to assess the role of the CSP in the protection conferred by vaccination with radiation-attenuated P. berghei parasites. Our data demonstrated that sterile immunity could be obtained despite the absence of immune responses specific to the CSP expressed by the parasite used for challenge., Conclusions: We conclude that other pre-erythrocytic parasite antigens, possibly hitherto uncharacterised, can be targeted to induce sterile immunity against malaria. From a broader perspective, our results raise the question as to whether immunodominant parasite antigens should be the favoured targets for vaccine development.

The review surveys potential "structural antigens" which represent small protein domains that can be chemically synthesized and, isolated from the context of the whole protein, can fold in the same native structure. They include natively unfolded protein regions, small globular domains, alpha-helical coiled coils and regions with tandem repeats forming structures ranging from the collagen triple helices to solenoid-like arrangements. We also describe and compare new strategies for development of vaccine that use the concept of structural epitopes. One type of approach is based on engineering artificial mini-proteins able to mimic structural epitopes of natural proteins. The review compares the "engineering" methodologies with "bioinformatics" approaches that became possible recently, after the sequencing of the genomes of many pathogens, and involve genome-wide bioinformatics searches for "structural antigens". In particular, based on the known P. falciparum genome, we identified putative alpha-helical coiled coil regions, 30-40 amino acids long, in proteins presented in asexual malaria blood stages. Peptides of such regions frequently fold into the "native" structure. A hundred such peptides were synthesized and all of them were recognized at various degrees (5-80%) by a panel of sera from donors living in malaria-endemic areas. The results obtained demonstrate that a bioinformatics/chemical synthesis strategy can rapidly lead to the identification of new proteins that can be targets of potential vaccines and/or drugs against malaria and other infectious organisms.

Both IFN-gamma and IL-12 play critical roles in defence against malaria. In a previous study, using Plasmodium yoelii model, C57BL/6 IFN-gamma receptor deficient mice (IFN-gammaR-/-) failed to develop protective immunity after a single immunization with irradiated sporozoites, but were protected after multiple immunizations. In contrast, in another study, BALB/c IFN-gamma gene knockout mice (IFN-gamma-/-) and BALB/c IL-12-deficient mice (IL-12p40-/- and IL-12p35-/-) were unable to mount protective immune response even after multiple immunizations with the same irradiated parasites. To better define the role of IFN-gamma and IL-12p40 in sterile protection, we selected the C57BL/6 model. Wild-type and IL-12p40-/- mice were immunized with a single or multiple doses of P. berghei irradiated sporozoites. While the wild-type mice were able to rapidly produce IFN-gamma and mount a protective immune response after a single immunization with irradiated sporozoites, IL-12p40-/- mice were neither able to produce IFN-gamma nor were protected. However, both strains of mice were able to produce IFN-gamma and were protected after three doses of irradiated sporozoites. Protection was partially and largely mediated by CD4+ T cells and CD8+ T cells, respectively. Thus, IL-12p40 plays an important role in mediating early protection by irradiated sporozoite immunization but is dispensable for protective immunity induced by several immunizations with irradiated sporozoites. Moreover, treatment of hyperimmune IL-12p40-/- mice with rhIL-18 bp-Fc, an inhibitor of IL-18 activity, did not abrogate protection indicating that IL-18 is not required for the effector phase of the immune response; it remains possible, however, that IL-18 may compensate for the lack of IL-12p40 in the induction phase of the immune response.

To identify malaria antigens for vaccine development, we selected alpha-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally "native" epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high alpha-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens.

The thrombospondin related adhesion protein (TRAP) is a malaria pre-erythrocytic antigen currently pursued as malaria vaccine candidate to Plasmodium falciparum. In this study, a long synthetic peptide (LSP) representing a P. vivax TRAP fragment involved in hepatocyte invasion was formulated in both Freund and Montanide ISA 720 adjutants and administered by IM and subcutaneous routes to BALB/c mice and Aotus monkeys. We measured specific humoral immune responses in both animal species and performed a sporozoite challenge in Aotus monkeys to assess the protective efficacy of the vaccine. After immunization both mice and Aotus seroconverted as shown by ELISA, and the specific anti-peptide antibodies cross reacted with the parasite in IFAT assays. Only two out of six immunized animals became infected after P. vivax sporozoite challenge as compared with four out of six animals from the control group. These results suggest that this TRAP fragment has protective potential against P. vivax malaria and deserves further studies as vaccine candidate.

A substantial and protective response against malaria liver stages is directed against the circumsporozoite protein (CSP) and involves induction of CD8(+) T cells and production of IFN-gamma. CSP-derived peptides have been shown to be presented on the surface of infected hepatocytes in the context of MHC class I molecules. However, little is known about how the CSP and other sporozoite Ags are processed and presented to CD8(+) T cells. We investigated how primary hepatocytes from BALB/c mice process the CSP of Plasmodium berghei after live sporozoite infection and present CSP-derived peptides to specific H-2K(d)-restricted CD8(+) T cells in vitro. Using both wild-type and spect(-/-) P. berghei sporozoites, we show that both infected and traversed primary hepatocytes process and present the CSP. The processing and presentation pathway was found to involve the proteasome, Ag transport through a postendoplasmic reticulum compartment, and aspartic proteases. Thus, it can be hypothesized that infected hepatocytes can contribute in vivo to the elicitation and expansion of a T cell response.

A brief historical account of synthetic peptide approach to malaria vaccine discovery and development, and recent accomplishments is presented. Application of this approach to high-throughput antigen discovery using the genome information is described. The need of a truly global effort and cooperation to defeat malaria is also discussed.

The glutamate-rich protein (GLURP) of P. falciparum is the target of cytophilic antibodies which are significantly associated with protection against clinical malaria. A phase 1 clinical trial was conducted in healthy adult volunteers with the long synthetic peptide (LSP) GLURP(85-213) combined with either Aluminum Hydroxide (Alum, 18 volunteers) or Montanide ISA 720 (ISA, 18 volunteers) as adjuvants. Immunizations with 10, 30 or 100 microg GLURP(85-213) were administered subcutaneously at days 0, 30, and 120. Adverse events occurred more frequently with increasing dosage of GLURP(85-213) LSP and were more prevalent in the ISA group. Serious vaccine-related adverse events were not observed. The vaccine induced dose-dependent cellular and humoral immune responses, with high levels of (mainly cytophilic IgG1) antibodies that recognize parasites by immunofluorescence (IFA). Plasma samples collected 30 days after the last immunization induced a dose-dependent inhibition of parasite growth in vitro in the presence of monocytes. In conclusion, immunizations with GLURP(85-213) LSP formulations induce adverse events but can be administered safely, generating antibodies with capacity to mediate growth-inhibitory activity against P. falciparum in vitro.

Although Plasmodium falciparum is the leading cause of morbidity and mortality due to malaria worldwide, nearly 2.5 billion people, mostly outside Africa, are also at risk from malaria caused by Plasmodium vivax infection. Currently, almost all efforts to develop a malaria vaccine have focused on P. falciparum. For example, there are 23 P. falciparum vaccine candidates undergoing advanced clinical studies and only two P. vivax vaccine candidates being tested in preliminary (Phase I) clinical trials, with few others being assessed in preclinical studies. More investment and a greater effort toward the development of P. vivax vaccine components for a multi-species vaccine are required. This is mainly because of the wide geographical coexistence of both parasite species but also because of increasing drug resistance, recent observations of severe and lethal P. vivax cases and relapsing parasite behaviour. Availability of the P. vivax genome has contributed to antigen discovery but new means to test vaccines in future trials remain to be designed.

The purpose of this work was to test the immunogenicity in C57BL mice of two synthetic peptides derived from the constant region of 3D7 and FC27 Plasmodium falciparum MSP2 dimorphic proteins, either microencapsulated into poly-lactide-co-glycolide acid microparticles (PLGA MP) or delivered with the human compatible adjuvant Montanide ISA 720 for comparison. Potent and prolonged antibody responses were obtained for both peptides by using PLGA MP formulations after subcutaneous or intradermal injections. As compared to the subcutaneous route of immunization, the intradermal route induced greater immune responses. Montanide adjuvant was effective in eliciting antibodies against the 3D7 peptide but not against the FC27 peptide. Peptide-specific cytophilic antibodies (IgG2a) were detected after boosting with homologous peptide for all vaccine formulations. MP formulations elicited a lower IgE secretion as compared to that observed for both Montanide formulated vaccines. Our results demonstrate the ability of the polymer microparticles to overcome the lack of immunogenicity of FC27 MSP2 peptide in C57BL mice and their potential to induce desirable immune responses against malaria.

Purpose: To redirect an ongoing antiviral T-cell response against tumor cells in vivo, we evaluated conjugates consisting of antitumor antibody fragments coupled to class I MHC molecules loaded with immunodominant viral peptides., Experimental Design: First, lymphochoriomeningitis virus (LCMV)-infected C57BL/6 mice were s.c. grafted on the right flank with carcinoembryonic antigen (CEA)-transfected MC38 colon carcinoma cells precoated with anti-CEA x H-2D(b)/GP33 LCMV peptide conjugate and on the left flank with the same cells precoated with control anti-CEA F(ab')(2) fragments. Second, influenza virus-infected mice were injected i.v., to induce lung metastases, with HER2-transfected B16F10 cells, coated with either anti-HER2 x H-2D(b)/NP366 influenza peptide conjugates, or anti-HER2 F(ab')(2) fragments alone, or intact anti-HER2 monoclonal antibody. Third, systemic injections of anti-CEA x H-2D(b) conjugates with covalently cross-linked GP33 peptides were tested for the growth inhibition of MC38-CEA(+) cells, s.c. grafted in LCMV-infected mice., Results: In the LCMV-infected mice, five of the six grafts with conjugate-precoated MC38-CEA(+) cells did not develop into tumors, whereas all grafts with F(ab')(2)-precoated MC38-CEA(+) cells did so (P = 0.0022). In influenza virus-infected mice, the group injected with cells precoated with specific conjugate had seven times less lung metastases than control groups (P = 0.0022 and P = 0.013). Most importantly, systemic injection in LCMV-infected mice of anti-CEA x H-2D(b)/cross-linked GP33 conjugates completely abolished tumor growth in four of five mice, whereas the same tumor grew in all five control mice (P = 0.016)., Conclusion: The results show that a physiologic T-cell antiviral response in immunocompetent mice can be redirected against tumor cells by the use of antitumor antibody x MHC/viral peptide conjugates.

Little is known about the role of conformation on the antigen processing by antigen presenting cells. Using a well-defined antigen containing two disulfide bridges, the synthetic C-terminal fragment 282-383 derived from Plasmodium falciparum circumsporozoite protein (PfCS 282-383), we show that the reduced form is presented in vitro more efficiently than its oxidized counterpart, inducing stronger CTL recognition. In addition, only the reduced form can be presented by the TAP independent T2 cell line. Thus, the reduced form is processed by TAP dependent and independent pathways.

Integrin receptors are the main mediators of cell adhesion to the extracellular matrix. They bind to their ligands by interacting with short amino acid sequences, such as the RGD sequence. Soluble, small RGD-based peptides have been used to block integrin-binding to ligands, thereby interfering with cell adhesion, migration and survival, while substrate-immobilized RGD sequences have been used to enhance cell binding to artificial surfaces. This approach has several important medical applications, e.g. in suppression of tumor angiogenesis or stimulation of bone formation around implants. However, the relatively weak affinity of short RGD-containing peptides often results in incomplete integrin inhibition or ineffective ligation. In this work, we designed and synthesized several new multivalent RGD-containing molecules and tested their ability to inhibit or to promote integrin-dependent cell adhesion when used in solution or immobilized on substrates, respectively. These molecules consist of an oligomeric structure formed by alpha-helical coiled coil peptides fused at their amino-terminal ends with an RGD-containing fragment. When immobilized on a substrate, these peptides specifically promoted integrin alphaVbeta3-dependent cell adhesion, but when used in solution, they blocked alphaVbeta3-dependent cell adhesion to the natural substrates fibronectin and vitronectin. One of the peptides was nearly 10-fold more efficient than fibronectin or vitronectin in promoting cell adhesion, and almost 100-fold more efficient than a linear RGD tripeptide in blocking adhesion. These results indicate that alpha-helical coiled coil peptides carrying an amino-terminal RGD motif can be used as soluble antagonists or surface-immobilized agonists to efficiently inhibit or promote integrin alphaVbeta3-mediated cell adhesion, respectively., (Copyright (c) 2005 European Peptide Society and John Wiley & Sons, Ltd.)

The purpose of this study was to analyze the potential of various types of biodegradable microspheres (MS) (i) to activate in vitro cell line-derived macrophages (RAW 264.7, Mphi), and primary peritoneal and bone marrow-derived mouse Mphi, to prolong the release and presentation of microencapsulated synthetic malaria antigens by Mphi after uptake of antigen-loaded MS, and (ii) to stimulate an immune response in mice against a microencapsulated synthetic malaria antigen. The MS were made of various types of poly(lactide-co-glycolide) (PLGA) or chitosan cross-linked with tripolyphosphate. PLGA, but not chitosan MS, were efficiently ingested by Mphi. Upon exposure to the various MS types, Mphi increased only the production of reactive oxygen intermediates (ROI), while the production of nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), and the expression of cyclooxigenase-2 (COX-2), inducible NO synthase (iNOS), the cell surface markers MHC class I and II, and CD 86 remained unaffected. In vitro release of the microencapsulated antigen from PLGA50:50 MS followed a pulsatile pattern and extended over 14 weeks. This prolonged antigen release was also mirrored in the significantly prolonged antigen presentation over more than 7 days by Mphi after uptake of antigen-loaded PLGA MS. Finally, antigen-loaded PLGA MS induced a solid immune response in mice after a single s.c.-injection, which was only slightly inferior to the antibody titers measured with the control formulation with Montanide ISA720. These results suggest that MS are well tolerated by Mphi. The prolonged antigen presentation by Mphi, as measured in vitro, along with the capacity to induce a strong immune response in animals emphasize that biodegradable MS are a very promising delivery system for both preventive and immunotherapeutic vaccines.

The C-terminal conserved region of Plasmodium falciparum merozoite surface protein 3 (MSP3) is the trigger antigen of a protective immune response mediated by cytophilic antibodies. In an open, randomized, two-adjuvant (Montanide ISA 720, aluminum hydroxide) phase I clinical trial we evaluated the safety and immunogenicity of increasing doses of a long synthetic peptide construct spanning the conserved region of MSP3 targeted by biologically active antibodies (MSP3-LSP). Thirty-five healthy volunteers were randomized to receive three subcutaneous injections on days 0, 30, and 120. Of the 100 injections given, 10 caused severe local reactions, 62 caused transient mild to moderate local reactions, and 28 caused no reaction. On the basis of preestablished exclusion criteria, use of the Montanide formulation led to withdrawal of five volunteers after the second injection. This led to a reduction in the subsequent vaccine doses in four of the groups. No vaccine-related serious adverse events occurred throughout the trial. After the third injection, volunteers displayed a marked specific anti-MSP3-LSP antibody response (23/30 individuals, compared with 29/34 individuals for plasma from an area where malaria is endemic), an anti-native MSP3 antibody response (19/30 individuals), a T-cell-antigen-specific proliferative response (26/30 individuals), and gamma interferon production (25/30 individuals). In conclusion, the MSP3-LSP vaccine was immunogenic with both adjuvants, although it was unacceptably reactogenic when it was combined with Montanide. The potential usefulness of the candidate vaccine is supported by the induction of a strong cytophilic response (i.e., the type of anti-MSP3 antibodies involved in antibody-dependent, monocyte-mediated protective mechanisms in areas where malaria is endemic).

Background: Plasmodium falciparum merozoite surface protein 3 is a malaria vaccine candidate that was identified, characterised, and developed based on a unique immuno-clinical approach. The vaccine construct was derived from regions fully conserved among various strains and containing B cell epitopes targeted by human antibodies (from malaria-immune adults) that are able to mediate a monocyte-dependent parasite killing effect. The corresponding long synthetic peptide was administered to 36 volunteers, with either alum or Montanide ISA720 as adjuvant., Methods and Findings: Both formulations induced cellular and humoral immune responses. With alum, the responses lasted up to 12 mo. The vaccine-induced antibodies were predominantly of cytophilic classes, i.e., able to cooperate with effector cells. In vitro, the antibodies induced an inhibition of the P. falciparum erythrocytic growth in a monocyte-dependent manner, which was in most instances as high as or greater than that induced by natural antibodies from immune African adults. In vivo transfer of the volunteers' sera into P. falciparum-infected humanized SCID mice profoundly reduced or abrogated parasitaemia. These inhibitory effects were related to the antibody reactivity with the parasite native protein, which was seen in 60% of the volunteers, and remained in samples taken 12 mo postimmunisation., Conclusion: This is the first malaria vaccine clinical trial to clearly demonstrate antiparasitic activity by vaccine-induced antibodies by both in vitro and in vivo methods. The results, showing the induction of long-lasting antibodies directed to a fully conserved polypeptide, also challenge current concepts about malaria vaccines, such as unavoidable polymorphism, low antigenicity, and poor induction of immune memory.

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.

Stable protective immunity can be achieved against malaria by the injection of radiation-attenuated sporozoites (gamma-spz) and is mediated by IFN-gamma producing CD8+ T cells targeting the pre-erythrocytic stages. An efficient malaria vaccine should mimic this immunity. We compared the immune response specific for the circumsporozoite protein (CSP) of Plasmodium berghei (P. berghei), an important target of this protective response, elicited in mice immunized with the long synthetic polypeptide (LSP) PbCS 242-310, representing the C-terminus of the CSP of P. berghei, with the adjuvant QS-21 or injected with gamma-spz. The ex vivo evaluation of the CD8+ T cell response by IFN-gamma ELISPOT assay revealed that the injection of LSP with QS-21 induced, compared to gamma-spz, a similar frequency of peptide-specific lymphocytes in the spleen but a eight-fold increase in the draining lymph-nodes. A very high frequency of CD8+ T cells, specific for the sequence PbCS 245-253, a H-2Kd-restricted CTL epitope, was obtained in the liver and spleen of mice immunized with the two regimens. Even though the frequency of H-2Kd PbCS 245-253 multimer+, CD8+ T cells was higher in gamma-spz immunized mice, the frequency of IFN-gamma producing CD8+ T cells was comparable. The phenotype of the CD8+ T cell responses was characterized with the help H-2Kd PbCS 245-253 multimer and most of the CSP-specific CD8+ T cells represented an intermediate subset between effector and central memory with CD44(high), CD45RB(high), CD62L(low) and CD122(high). The number of memory CD8+ T cells decreased after the last LSP immunization but could be boosted to higher level with live spz. The unique combination of LSP PbCS 242-310 and the adjuvant QS-21 induced an immune response that was comparable in terms of quality to the one generated with gamma-spz. This confirmed the potential of LSP as malaria vaccine candidates as well as for the study of the repertoire of targets of protective immunity in the gamma-spz vaccine model.

The circumsporozoite (CS) is the most abundant surface protein of the Plasmodium sporozoite, and is also present early in the liver stage of the infection. Following successful protective experiments in mice and monkeys, the synthetic 102-mer malaria vaccine polypeptide representing the C-terminal region of the CS of Plasmodium falciparum was tested in a clinical trial with healthy human volunteers. This vaccine induced strong CD8(+), CD4(+) T lymphocyte and antibody responses specific for the immunizing peptide. CD8(+) T lymphocyte responses elicited in HLA-A*0201 volunteers recognized two well-defined cytotoxic T lymphocyte epitopes within the CS. Here, we show that both monocyte-derived dendritic cells (Mo-DC) and Epstein-Barr virus-transformed B-lymphoblastoid cells (LCL) can present a cytotoxic T lymphocyte epitope contained within the 102-mer synthetic peptide. Paraformaldehyde and low temperature inhibited presentation, indicating that cellular processing was required. Using specific inhibitors, we show that, in both cell types, processing requires the proteasome and the MHC class I pathway, while the endosomal compartment appears to be critical only for the presentation by Mo-DC. Antigen uptake is associated with actin polymerization in both cell types. These in vitro results demonstrate the likely pathway of antigen presentation achieved via vaccination with this synthetic peptide.

Plasmodium falciparum MSP6 is a merozoite surface antigen that shows organization and sequence homologies similar to those of MSP3. Within its C-terminus conserved region, it presents some epitopes that are cross-reactive with MSP3 and others that are not, both being targets of naturally occurring antibodies that block the P. falciparum erythrocytic cycle in cooperation with monocytes.

Three long synthetic peptides corresponding to amino (N), repeat (R) and carboxyl (C) regions of the Plasmodium vivax circumsporozoite (CS) protein were synthesised and used to assess their potential as vaccine candidates. Antigenicity studies were carried out using human blood samples from residents of a malaria-endemic area of Colombia, and immunogenicity was tested in Aotus monkeys. The N and C peptides spanned the total native amino and carboxyl flanking regions, whereas the R peptide corresponded to a construct based on the first central nona-peptide repeated in tandem three times and colinearly linked to a universal T-cell epitope (ptt-30) derived from tetanus toxin. All three peptides had been shown previously to contain several B-, T-helper (Th) and Cytotoxic T Lymphocytes (CTL) epitopes. Sixty-one percent of the human sera reacted with the R region, whereas 35 and 39% of the samples had antibodies against the N and C peptides, respectively. Human Peripheral Blood Mononuclear Cells (PBMC) showed higher levels of IFN-gamma than IL-4 when stimulated with peptides containing Th epitopes. Aotus monkeys immunised with the peptides formulated in either Montanide ISA720 or Freund's adjuvants produced strong antibody responses that recognised the peptide immunogens and the native circumsporozoite protein on sporozoites. Additionally, high IFN-gamma production was induced when Aotus lymphocytes were stimulated in vitro with each of the three peptides. We observed boosting of antibody responses and IFN-gamma production by exposure to live sporozoites. These results confirm the high antigenicity and immunogenicity of such synthetic polypeptides and underline their vaccine potential.

One potential benefit of DNA vaccines is the capacity to elicit antibody and T-cell responses against multiple antigens at the same time by mixing plasmids expressing different proteins. A possible negative effect of such mixing is interference among plasmids regarding immunogenicity. In preparation for a clinical trial, we assessed the immunogenicity of GMP-produced plasmids encoding five Plasmodium falciparum proteins, PfCSP, PfSSP2, PfEXP1, PfLSA1, and PfLSA3, given as a mixture, or alone. The mixture induced higher levels of antibodies against whole parasites than did the individual plasmids, but was associated with a decrease in antibodies to individual P. falciparum proteins. T-cell responses were in general decreased by administration of the mixture. Immune responses to individual plasmids and mixtures were generally higher in inbred mice than in outbreds. In inbred BALB/c and C57BL/6 mice, coadministration of a plasmid expressing murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), increased antibody and T-cell responses, but in outbred CD-1 mice, coadministration of mGM-CSF was associated with a decrease in antibody responses. Such variability in data from studies in different strains of mice underscores the importance of genetic background on immune response and carefully considering the goals of any preclinical studies of vaccine mixtures planned for human trials.

Biodegradable microspheres may represent a potential tool for the delivery of combination vaccines. We demonstrate strong immunogenicity of five co-encapsulated antigens after a single subcutaneous inoculation in guinea pigs. Tetanus- and diphtheria-specific antibodies were not significantly affected by the presence of either antigen or by the presence of pertussis or Haemophilus influenzae type b (Hib) antigens. Microsphere formulations gave better protection against diphtheria toxin than did two injections of a licensed tetravalent vaccine. Finally, a synthetic malaria peptide antigen (PfCS) also encapsulated in PLGA microspheres increased diphtheria and tetanus-specific immunity and improved protection against diphtheria. These findings demonstrate the potential of microspheres as an alternative and promising strategy for combination vaccines with a further aptitude in reducing the number of inoculations required to gain functional immunity.

This review covers the latest developments of long synthetic peptide technology for the rapid identification and development of malaria vaccine candidates and immunological modulators. A brief description of the two most common solid-phase synthetic procedures, together with the latest advances in optimisation of peptide chain assembly and analytical instrumentation, is given, with special attention to non-specialists. Several examples of vaccine candidates developed in the authors' or their collaborators' laboratories are also provided.

Merozoite surface protein 3 (MSP3) is a target of antibody-dependent cellular inhibition (ADCI), a protective mechanism against Plasmodium falciparum malaria. From the C-terminal half of the molecule, 6 overlapping peptides were chosen to characterize human immune responses. Each peptide defined at least 1 non-cross-reactive B cell epitope. Distinct patterns of antibody responses, by level and IgG subclass distribution, were observed in inhabitants of a malaria-endemic area. Antibodies affinity purified toward each peptide differed in their functional capacity to mediate parasite killing in ADCI assays: 3 of 6 overlapping peptides had a major inhibitory effect on parasite growth. This result was confirmed by the passive transfer of anti-MSP3 antibodies in vivo in a P. falciparum mouse model. T helper cell epitopes were identified in each peptide. Antigenicity and functional assays identified a 70-amino acid conserved domain of MSP3 as a target of biologically active antibodies to be included in future vaccine constructs based on MSP3.

The recent availability of significantly increased levels of funding for unmet medical needs in the developing world, made available by newly created public-private-partnerships, has proven to be a powerful driver for stimulating clinical development of candidate vaccines for malaria. This new way forward promises to greatly increase the likelihood of bringing a safe and effective vaccine to licensure. The investigators bring together important published and unpublished information that illuminates the status of malaria vaccine development. They focus their comments on those candidate vaccines that are currently in or expected to enter clinical trials in the next 12 months., (Copyright 2004 The American Society of Tropical Medicine and Hygiene)

A longitudinal study was undertaken in Burkina Faso among 293 children aged 6 months to 9 years in order to determine the correlation between an antibody response to several individual malarial antigens and malarial infection. It was found that the presence of a positive antibody response at the beginning of the rainy season to three long synthetic peptides corresponding to Plasmodium falciparum Exp-1 101-162, MSP-3 154-249 and GLURP 801-920 but not to CSP 274-375 correlated with a statistically significant decrease in malarial infection during the ongoing transmission season. The simultaneous presence of an antibody response to more than one antigen is indicative of a lower frequency of malarial infection. This gives scientific credibility to the notion that a successful malaria vaccine should contain multiple antigens.

Self-assembling peptides present attractive platforms for engineering materials with controlled nanostructures. Recently, an alpha-helical fibril forming peptide (alphaFFP) was designed that self-assembles into nanofibrils at acid pH. Circular dichroism spectroscopy, electron-microscopy and x-ray fibre diffraction data showed that the most likely structure of alphaFFP fibrils is a five-stranded coiled coil rope. In the present study, scanning transmission electron microscopy (STEM) was used to improve our understanding of the alphaFFP fibril structure. The measurements of fibril mass per length suggest that there are ten alpha-helices in transverse sections of the fibrils. Based on the known data, it is proposed that a predominant fibrillar structure of alphaFFP is a dimer of alpha-helical five stranded protofilaments wrapped around a common axis. It is shown that these structures have an axial dimension of 58 +/- 16 nm and a width of 4 +/- 1 nm. A small number of thin fibrils is also observed in the negative stained preparation and STEM images. The thin fibrils may correspond to the single protofilament.

Background: Nasal administration of major peptide T cell epitopes gives contradictory data on the induction of peripheral tolerance., Objective: To compare the prophylactic effect of intranasal treatment (INT) on the development of an allergic response, using either ovalbumin (OVA) or its major T cell epitope OVA 323-339 (OVAp)., Methods: BALB/c mice were treated intranasally with OVA or OVAp and subsequently immunized s.c. with OVA. Anti-OVA-specific antibody, T cell proliferation and cytokine responses were analysed. In an adoptive transfer model using OVAp specific TCR transgenic (Tg) T cells from D011.10 mice, in vivo tracking and characterization of transferred T cells in the cervical, inguinal and bronchial lymph nodes (BLN) and in the spleen were determined by FACS analysis., Results: Prophylactic INT with OVA induced T cell tolerance towards subsequent OVA s.c. immunizations, inhibiting OVA specific T cell proliferation, IgE and IgG1 production, in contrast to INT with OVAp, which was unable to induce tolerance. In vivo analysis of transferred OVA-specific TCR Tg T cells showed that INT with OVA induced a preferential activation of T cells in BLN, as opposed to a broad, systemic activation with OVAp. In vivo, OVAp INT led to faster and more sustained cell division cycles than OVA INT. Ex vivo, tolerance to OVA was associated with the generation of IL-10 secreting CD4(+) T cells in BLN of OVA-treated mice only., Conclusion: INT with OVA but not with OVAp led to regional (as opposed to systemic) T cell activation and the induction of IL-10 secreting CD4(+) T cells in BLN, potentially critical steps in the induction of T cell-specific tolerance via the nasal route.

Recently, we designed a short alpha-helical fibril-forming peptide (alphaFFP) that can form alpha-helical nanofibrils at acid pH. The non-physiological conditions of the fibril formation hamper biomedical application of alphaFFP. It was hypothesized that electrostatic repulsion between glutamic acid residues present at positions (g) of the alphaFFP coiled-coil sequence prevent the fibrillogenesis at neutral pH, while their protonation below pH 5.5 triggers axial growth of the fibril. To test this hypothesis, we synthesized alphaFFPs where all glutamic acid residues were substituted by glutamines or serines. The electron microscopy study confirmed that the modified alphaFFPs form nanofibrils in a wider range of pH (2.5-11). Circular dichroism spectroscopy, sedimentation, diffusion and differential scanning calorimetry showed that the fibrils are alpha-helical and have elongated and highly stable cooperative tertiary structures. This work leads to a better understanding of interactions that control the fibrillogenesis of the alphaFFPs and opens opportunities for their biomedical application.

Insecticide treated materials (ITM) are considered a useful malaria control measure for endemic countries, but whether they also delay the acquisition of immunity to malaria remains unclear. This study investigates plasma antibody levels in 160 children aged 3-6 years from five villages protected by insecticide treated curtains (ITC) over 6 years and in 184 children of the same age group from five villages in the same area never covered by ITC. The antigens to which antibodies were investigated were: the Plasmodium falciparum circumsporozoite protein (CSP) repetitive sequence (NANP)5; the C-terminal domain of the P. falciparum exported protein 1 (Cter-PfExp1); three fragments of the glutamate rich protein (GLURP), referred to as R0, R1 and R2; the merozoite surface protein 3 (MSP3). The level of antibodies was lower in children from the ITC area than in children from the non-ITC area for (NANP)5, R0, R2 and MSP3. Prevalence and intensity of P. falciparum infection were similar in the two groups of children. These findings suggest that reducing the level of malaria transmission over a long period may affect the level of antibodies in children to both sporozoite and blood stage malaria antigens.

The concept of antibody-mediated targeting of antigenic MHC/peptide complexes on tumor cells in order to sensitize them to T-lymphocyte cytotoxicity represents an attractive new immunotherapy strategy. In vitro experiments have shown that an antibody chemically conjugated or fused to monomeric MHC/peptide can be oligomerized on the surface of tumor cells, rendering them susceptible to efficient lysis by MHC-peptide restricted specific T-cell clones. However, this strategy has not yet been tested entirely in vivo in immunocompetent animals. To this aim, we took advantage of OT-1 mice which have a transgenic T-cell receptor specific for the ovalbumin (ova) immunodominant peptide (257-264) expressed in the context of the MHC class I H-2K(b). We prepared and characterized conjugates between the Fab' fragment from a high-affinity monoclonal antibody to carcinoembryonic antigen (CEA) and the H-2K(b) /ova peptide complex. First, we showed in OT-1 mice that the grafting and growth of a syngeneic colon carcinoma line transfected with CEA could be specifically inhibited by systemic injections of the conjugate. Next, using CEA transgenic C57BL/6 mice adoptively transferred with OT-1 spleen cells and immunized with ovalbumin, we demonstrated that systemic injections of the anti-CEA-H-2K(b) /ova conjugate could induce specific growth inhibition and regression of well-established, palpable subcutaneous grafts from the syngeneic CEA-transfected colon carcinoma line. These results, obtained in a well-characterized syngeneic carcinoma model, demonstrate that the antibody-MHC/peptide strategy can function in vivo. Further preclinical experimental studies, using an anti-viral T-cell response, will be performed before this new form of immunotherapy can be considered for clinical use.

The goal of this project was the evaluation of a novel immunomodulatory adjuvant for human use, OM-174, which is a soluble adjuvant derived from Escherichia coli lipid A. For this study, we used a synthetic peptide, known for its safety and reproducibility and the murine model of BALB/c mice. The long peptide (PbCS 242-310) used corresponds to the C-terminal region of the circumsporozoite protein (CSP) that is the major protein on the surface of Plasmodium sporozoites. Subcutaneous injections of PbCS 242-310 in combination with soluble adjuvant OM-174 induced long lasting peptide-specific antibody titres comparable to those obtained by immunization with incomplete Freund's adjuvant (IFA). The ex vivo evaluation of the CD8(+) T cell response by IFN-gamma ELISPOT assay revealed that the injection of polypeptide with OM-174 adjuvant induced, compared to IFA, a similar and an eight-fold increased frequency of peptide-specific lymphocytes in the draining lymph-nodes and in the spleen, respectively. The CD8(+) T-cells are specific for the sequence PbCS 245-253, a well-known H-2K(d)-restricted CTL epitope, and are cytotoxic as shown in a chromium release assay. Immunization of BALB/c mice with this polypeptide in combination with adjuvant OM-174 conferred a protection after challenge with live Plasmodium berghei sporozoites.The strong antibody and CTL responses observed to a synthetic peptide in mice, the safety profile of the adjuvant and its extensive physico-chemical characterization suggest that OM-174 has a potential use in vaccine formulations for humans.

Background: There is a need to improve the safety and efficacy of allergen-specific immunotherapy. Long synthetic peptide-based immunotherapy was proven safe, immunogenic, and protective in preclinical trials., Objective: To evaluate the safety and immunogenicity of an allergen-derived long synthetic overlapping peptide (LSP) immunotherapy, we designed a double-blind, placebo-controlled phase I clinical trial in patients hypersensitive to bee venom., Methods: Patients from the active group were injected at day 0 with a mixture of 3 LSPs mapping the entire PLA2 molecule, a major bee venom allergen, in a dose-escalating protocol to a maintenance dose of 100 microg per peptide repeated at days 4, 7, 14, 42, and 70. The control group was injected with human albumin., Results: Whereas specific T-cell proliferation in the peptide group increased up to day 14, a sharp decline was observed thereafter, ending in specific T-cell hyporesponsiveness at day 80. Serum-specific IgG4 response was enhanced, in contrast to anti-PLA2 IgE. Specific T-cell cytokine modulation was marked by increased IL-10 and IFN-gamma secretion. LSP injections were well tolerated in all patients except for mild, late allergic reactions in 2 patients at day 70., Conclusions: The results of this short-term study demonstrate that LSP-based allergen immunotherapy was safe and able to induce T(H)1-type immune deviation, allergen-specific IL-10 production, and T-cell hyporesponsiveness. LSPs, which offer the advantage of covering all possible T-cell epitopes for any HLA genotype, can be considered candidates for a novel and safe approach of specific immunotherapy.

Biodegradable microspheres (MS) consisting of poly(D,L-lactide-co-glycolide) (PLGA) represent a promising alternative to conventional adjuvants. The adjustable pulsatile release of encapsulated material from such MS offers the potential to mimic the priming and boosting injections of conventional immunization regimens. In this paper, we demonstrate that MS can serve as antigen reservoirs in antigen presenting cells (APC), so that antigen is presented for extended periods of time (up to 9 days). In particular, we could show by measurement of IFN-gamma production that encapsulated peptides were presented to cytotoxic T lymphocytes (CTL) by mouse and human macrophages as well as by human dendritic cells in vitro for a longer time period as compared to soluble peptides. The extended antigen presentation may thus improve the CTL response in vivo. These results may be of paramount importance in cancer vaccination therapy since MS may serve as antigen reservoirs to extend the presentation time by APC used to boost the patient's immune response to tumor antigens., (Copyright 2002 Elsevier Science Ltd.)

The zinc metalloprotease gp63 (leishmanolysin; promastigote surface protease) is expressed at high density at the surface of Leishmania promastigotes. Efficient non-toxic inhibitors of gp63 do not exist, and its precise role in parasite physiology remains unknown. MARCKS (myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in various cells, including macrophages. We reported previously that MRP is an excellent substrate for gp63. A major cleavage site was identified within the MRP effector domain (ED), a highly basic 24-amino-acid sequence, and the synthetic ED peptide (MRP(ED)) was shown to inhibit MRP hydrolysis. In the present study, MRP cleavage was used as an assay to measure the capacity of various MRP or MARCKS ED peptides to block gp63 activity. On a molar basis, MRP(ED) inhibited gp63 to a greater extent than two previously described gp63 inhibitors, o -phenanthroline and benzyloxycarbonyl-Tyr-Leu-NHOH. MARCKS(ED) analogues containing modifications in the gp63 consensus cleavage site showed significant differences in inhibitory capacity. As phosphorylation of ED serine residues prevented gp63-mediated MRP degradation, we synthesized a pseudophosphorylated peptide in which serine residues were substituted by aspartate (3DMRP(ED)). 3DMRP(ED) was a highly effective inhibitor of both soluble and parasite-associated gp63. Finally, MRP ED peptides were synthesized together with an N-terminal HIV-1 Tat transduction domain (TD) to obtain cell-permeant peptide constructs. Such peptides retained gp63 inhibitory activity and efficiently entered both macrophages and parasites in a Tat TD-dependent manner. These studies may provide the basis for developing potent cell-permeant inhibitors of gp63.

The demonstration of the i) acquired protective immunity in adults living in endemic areas, ii) cure of malaria patients with passive transfer of specific immunoglobulins, and iii) protection conferred by vaccination with sporozoites attenuated by radiation, justifies the search for a malaria vaccine. Given the improbability that a vaccine directed against a single antigen will be completely protective, the preferred option is to combine several antigens of different stages of the parasite in a multi-component multi-stage vaccine which is likely to protect both the travellers and the populations living in endemic areas. Potential manufacturing technologies include recombinant proteins, synthetic peptides and DNA vaccines, the relevant genes encoding malaria antigens being inserted into a plasmid or a live vector such as vaccinia or poxvirus. A number of human trials using different antigens and technologies have been carried out in the last ten years. Three vaccines have undergone safety and efficacy testing in the field. SPf66, comprising a linear polymerised synthetic peptide with several distinct epitopes, has been extensively evaluated in different epidemiological settings. The efficacy overall was 23%, but was only 2% in African infants, the most susceptible group. The circumsporozoite recombinant protein fused with the antigen S of the hepatitis B virus and formulated in a potent adjuvant (RTS,S) led to a high, but short-term, level of protection against infection and disease in Gambian adults. The first pure asexual blood-stage vaccine comprising three antigens of the merozoite stage (MSP1&2 and RESA, Combination B) had an efficacy of 62% in reducing parasite density in Papua New Guinean children. A malaria vaccine that can reduce the burden of disease in the most affected populations is thus an achievable goal, and each trial provides additional knowledge about mechanisms of protection as well as about new vaccine technology.

The injection of a mixture of bona fide T cell epitopes can lead to the occurrence of immunodominance, meaning that the immune response is focused on the recognition of a single epitope or a small portion of the epitopes injected. We have previously demonstrated that the administration of rIL-12 can counteract immunodominance in BALB/c mice. In this study, we show that the administration of rIL-12 to HLA-A2.1 transgenic mice (A2k(b) mice) abrogates specifically the immune response against HLA-A2.1-restricted HIV epitopes in the spleen. This lack of immune response is most probably due to a transient depletion of B cells, T cells, macrophages, and dendritic cells in this organ. Therefore, our study explains the mechanism of immunosuppression by rIL-12 in vivo.

Background: Murine models of hypersensitivity to allergens are useful tools for the evaluation of preclinical strategies to down-regulate the IgE response., Objective: To monitor the long-term kinetics of T and B cell responses to allergen as a function of allergen dosage and to investigate the effect of parallel immunization with a second antigen; to correlate B cell response with anaphylaxis., Methods: CBA/J mice were sensitized every other week by subcutaneous injections of phospholipase A2 (PLA2) and/or ovalbumin (OVA) adsorbed to alum. Specific antibody isotype responses, T cell proliferation, T cell cytokine production and anaphylaxis were assessed throughout the sensitization phase., Results: Low-dose immunization with PLA2 (0.1 microg) favoured a long-term, specific T helper (Th)2 response with high IgE and IL-4 production in contrast to high-dose PLA2 (10 microg) immunization, which biased the immune response towards a Th1 response with high IgG2a and low IL-4 production. Parallel immunization with an unrelated antigen (ovalbumin) had a significant bystander effect on the immunization with PLA2, which was also dose-dependent. Finally, although anaphylaxis as measured by rectal temperature drop was allergen-specific, it could be induced in the high- and low-dose immunization groups, and was not solely dependent on IgE levels., Conclusion: Though low-dose allergen immunization appears to induce an efficient IgE response, the intensity and quality of this response may be modulated by bystander effects of parallel immunization and does not correlate strictly with anaphylaxis. This observation has relevance to the design of clinical immunotherapy protocols using murine model-based data.

Specific CD8(-) T-lymphocyte (CTL) activity against Plasmodium pre-erythrocytic stages (P-ES) derived antigens is considered one of the most important mechanisms for malaria protection. Plasmodium vivax is the second most prevalent human malaria parasite species distributed worldwide. Although several CTL epitopes have been identified in Plasmodium falciparum P-ES derived antigens, none has been described for P. vivax to date. In this study, we analysed HLA-A*0201 specific CD8(-) T-lymphocyte responses to the P. vivax circumsporozoite (CS) protein in both malaria exposed and non-exposed populations from the Colombian Pacific Coast. First, we analysed the prevalence of HLA-A2 allele in the study populations and found that approximately 38 of the individuals expressed this molecule and that 50 of them were HLA-A*0201. We then selected, on the P. vivax CS, five peptide sequences containing the HLA-A*0201 binding motifs and used the corresponding synthetic peptides to evaluate the CD8(-) T-lymphocyte interferon (IFN)-gamma response. Peripheral blood mononuclear cells from the HLA-A*0201 donors were in vitro stimulated with these peptides and IFN-gamma production was determined by an ELISPOT assay. Specific CD8(-) T-lymphocyte responses were detected for three peptides located in the C-terminal region of the protein. Specific responses to these peptides were also detected in several individuals expressing different HLA-A*02 subtypes. The potential of these peptides to induce specific cytolysis and that of long synthetic peptides comprising these epitopes as P. vivax malaria vaccine subunits are being studied.

The present work describes the recognition of three synthetic polypeptides encompassing the N- and C-terminal regions of the transmembrane Exp-1 protein of the parasite Plasmodium falciparum by plasma and peripheral blood mononuclear cells from naturally exposed individuals living in African endemic areas. The three polypeptides comprise the sequences 23-105, 73-162 and 101-162, and overlap at the transmembrane domain (73-105). Thus, they permitted characterization of the immune response specific to the N- and C-terminal domains in an independent fashion. Two different populations were evaluated, one in the village of Safo in Mali and the other in the villages of Somnaway, Kabortenga and Toussouktenga in Burkina Faso. Antibodies to the sequence 73-162 of Pf Exp-1 were found in 70% of adult Mali donors and in all of the donors tested from Burkina Faso. Strikingly, the N-terminal fragment Pf Exp-1 23-105 was only weakly recognized by a few donors. Evaluation of the T-cell response indicated that the peptide Pf Exp-1 23-105 was more potent than Pf Exp-1 73-162 in inducing a proliferative response. A correlation between peptide-specific interferon-gamma and interleukin-6 production and proliferation to peptide Pf Exp-1 23-105 was observed. Further studies are needed to evaluate this molecule as a vaccine candidate.