[Show abstract][Hide abstract] ABSTRACT: Clinical profiling of two components for a synthetic peptide-based virosomal malaria vaccine has yielded promising results, encouraging the search for additional components for inclusion in a final multi-valent vaccine formulation. This report describes the immunological characterization of linear and cyclized synthetic peptides comprising amino acids 211-237 of Plasmodium falciparum merozoite surface protein (MSP-3).
These peptides were coupled to phosphatidylethanolamine (PE); the conjugates were intercalated into immunopotentiating reconstituted influenza virosomes (IRIVs) and then used for immunizations in mice to evaluate their capacity to elicit P. falciparum cross-reactive antibodies.
While all MSP-3-derived peptides were able to elicit parasite-binding antibodies, stabilization of turn structures by cyclization had no immune-enhancing effect. Therefore, further pre-clinical profiling was focused on FB-12, a PE conjugate of the linear peptide. Consistent with the immunological results obtained in mice, all FB-12 immunized rabbits tested seroconverted and consistently elicited antibodies that interacted with blood stage parasites. It was observed that a dose of 50 microg was superior to a dose of 10 microg and that influenza pre-existing immunity improved the immunogenicity of FB-12 in rabbits. FB-12 production was successfully up-scaled and the immunogenicity of a vaccine formulation, produced according to the rules of Good Manufacturing Practice (GMP), was tested in mice and rabbits. All animals tested developed parasite-binding antibodies. Comparison of ELISA and IFA titers as well as the characterization of a panel of anti-FB-12 monoclonal antibodies indicated that at least the majority of antibodies specific for the virosomally formulated synthetic peptide were parasite cross-reactive.
These results reconfirm the suitability of IRIVs as a carrier/adjuvant system for the induction of strong humoral immune responses against a wide range of synthetic peptide antigens. The virosomal formulation of the FB-12 peptidomimetic is suitable for use in humans and represents a candidate component for a virosomal multi-valent malaria subunit vaccine.
[Show abstract][Hide abstract] ABSTRACT: Matrix metalloproteinase-19 (MMP19) affects cell proliferation, adhesion, and migration in vitro but its physiological role in vivo is poorly understood. To determine the function of MMP19, we generated mice deficient for MMP19 by disrupting the catalytic domain of mmp19 gene. Although MMP19-deficient mice do not show overt developmental and morphological abnormalities they display a distinct physiological phenotype. In a model of contact hypersensitivity (CHS) MMP19-deficient mice showed impaired T cell-mediated immune reaction that was characterized by limited influx of inflammatory cells, low proliferation of keratinocytes, and reduced number of activated CD8(+) T cells in draining lymph nodes. In the inflamed tissue, the low number of CD8(+) T cells in MMP19-deficient mice correlated with low amounts of proinflammatory cytokines, especially lymphotactin and interferon-inducible T cell alpha chemoattractant (I-TAC). Further analyses showed that T cell populations in the blood of immature, unsensitized mice were diminished and that this alteration originated from an altered maturation of thymocytes. In the thymus, thymocytes exhibited low proliferation rates and the number of CD4(+)CD8(+) double-positive cells was remarkably augmented. Based on the phenotype of MMP19-deficient mice we propose that MMP19 is an important factor in cutaneous immune responses and influences the development of T cells.
PLoS ONE 06/2008; 3(6):e2343. DOI:10.1371/journal.pone.0002343 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Presentation of synthetic peptides on immunopotentiating reconstituted influenza virosomes is a promising technology for subunit vaccine development. An optimized virosomally delivered peptide representing 5 NPNA repeats of P. falciparum circumsporozoite protein is highly immunogenic in mice. Antibodies against this peptide (UK-39) inhibit sporozoite invasion of human hepatocytes. A second peptide (AMA49-C1) based on domain III of apical membrane antigen 1, induces antibodies that inhibit blood-stage parasite growth in vitro. Here we show a detailed pre-clinical profiling of these virosomally formulated peptides alone and in combination in mice and rabbits. Two immunizations with virosomally formulated UK-39 or AMA49-C1 were enough to elicit high titers of parasite cross-reactive antibodies in both species. A low dose of 10 microg UK-39 was enough to induce maximal titers in rabbits. Higher doses of peptide did not increase antibody titers. In contrast, AMA49-C1 induced higher antibody titers with 25 and 50 microg peptide. Combination of UK-39 and AMA49- C1 on separate virosomes did not have any negative effect on anti-peptide antibody titers in mice or rabbits. No MHC restriction was observed in the development of humoral responses in outbred rabbits with different immunogenetic backgrounds. All vaccine formulations were safe in toxicity studies in rabbits and rats. Taken together, low amounts of synthetic peptides delivered on virosomes induced high antibody titers in mice and rabbits. Moreover, different peptides could be combined without interfering with individual anti-peptide responses, augmenting the value of this system for the development of a multivalent malaria vaccine.
Human vaccines 03/2008; 4(2):106-14. DOI:10.4161/hv.4.2.5188 · 3.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Serine repeat antigen-5 (SERA5) is a candidate antigen for inclusion into a malaria subunit vaccine. During merozoite release and reinvasion the 120 kDa SERA5 precursor protein (P120) is processed, and a complex consisting of an N-terminal 47 kDa (P47) and a C-terminal 18kDa (P18) processing product associates with the surface of merozoites. This complex is thought to be involved in merozoite invasion of and/or egress from host erythrocytes. Here we describe the synthesis and immunogenic properties of virosomally formulated synthetic phosphatidylethanolamine (PE)-peptide conjugates, incorporating amino acid sequence stretches from the N-terminus of Plasmodium falciparum SERA5. Choosing an appropriate sequence was crucial for the development of a peptide that elicited high titers of parasite cross-reactive antibodies in mice. Monoclonal antibodies (mAbs) raised against the optimized peptide FB-23 incorporating amino acids 57-94 of SERA5 bound to both P120 and to P47. Western blotting analysis proved for the first time the presence of SERA5 P47 in sporozoites. In immunofluorescence assays, the mAbs stained SERA5 in all its predicted localizations. The virosomal formulation of peptide FB-23 is suitable for use in humans and represents a candidate component for a multi-valent malaria subunit vaccine targeting both sporozoites and blood stage parasites.
[Show abstract][Hide abstract] ABSTRACT: Matrix metalloproteinase-19 (MMP-19), unlike other members of the MMP family, is expressed in basal keratinocytes of intact epidermis whereas keratinocytes in suprabasal and higher epidermal layers express this enzyme only during cutaneous disorders. As the activity of MMP-19 effects proliferation, migration, and adhesion of keratinocytes we examined whether transcription factors involved in keratinocyte differentiation repress the expression of MMP-19. Using luciferase reporter assays, POU transcription factors Tst-1 (Oct-6) and Skn-1a (Oct-11) markedly downregulated the activity of MMP-19 promoter in COS-7 cells and HaCaT keratinocytes. Tst-1 alone was able to inhibit 85% of the promoter activity. Skn-1a exhibited a weak inhibitory effect although it synergistically increased effects of Tst-1. HaCaT cells stably transfected with Tst-1 showed a strong decrease of activity of MMP-19 promoter that correlated with suppression of MMP-19, cytokeratin 14 and 5, decreased cell proliferation, and altered expression of involucrin and loricrin. The expression of MMP-9 was also significantly reduced in Tst-1 expressing keratinocytes. MMP-2 was substantially affected during its activation whereas the expression of MMP-28 was unchanged. Our results suggest that Tst-1 and Skn-1a regulate expression of MMPs in keratinocytes and effect both the expression and activation of these proteolytic enzymes.
[Show abstract][Hide abstract] ABSTRACT: The circumsporozoite protein (CSP) of Plasmodium falciparum is a leading candidate antigen for inclusion in a malaria subunit vaccine. We describe here the design of a conformationally constrained synthetic peptide, designated UK-39, which has structural and antigenic similarity to the NPNA-repeat region of native CSP. NMR studies on the antigen support the presence of helical turn-like structures within consecutive NPNA motifs in aqueous solution. Intramuscular delivery of UK-39 to mice and rabbits on the surface of reconstituted influenza virosomes elicited high titers of sporozoite crossreactive antibodies. Influenza virus proteins were crucially important for the immunostimulatory activity of the virosome-based antigen delivery system, as a liposomal formulation of UK-39 was not immunogenic. IgG antibodies elicited by UK-39 inhibited invasion of hepatocytes by P. falciparum sporozoites, but not by antigenically distinct P. yoelii sporozoites. Our approach to optimized virosome-formulated synthetic peptide vaccines should be generally applicable for other infectious and noninfectious diseases.
[Show abstract][Hide abstract] ABSTRACT: Peptides delivered on the surface of influenza virosomes have been shown to induce solid humoral immune responses in experimental animals. High titers of peptide-specific antibodies were also induced in a phase 1a clinical trial in volunteers immunized with virosomal formulations of two peptides derived from the circumsporozoite protein (CSP) and the apical membrane antigen 1 (AMA-1) of Plasmodium falciparum. The main objective of this study was to perform a detailed immunological and functional analysis of the CSP-specific antibodies elicited in this phase 1a trial.
46 healthy malaria-naïve adults were immunized with virosomal formulations of two peptide-phosphatidylethanolamine conjugates, one derived from the NANP repeat region of P. falciparum CSP (designated UK-39) the other from P. falciparum AMA-1 (designated AMA49-C1). The two antigens were delivered in two different concentrations, alone and in combination. One group was immunized with empty virosomes as control. In this report we show a detailed analysis of the antibody response against UK-39. Three vaccinations with a 10 microg dose of UK-39 induced high titers of sporozoite-binding antibodies in all volunteers. This IgG response was affinity maturated and long-lived. Co-administration of UK-39 and AMA49-C1 loaded virosomes did not interfere with the immunogenicity of UK-39. Purified total IgG from UK-39 immunized volunteers inhibited sporozoite migration and invasion of hepatocytes in vitro. Sporozoite inhibition closely correlated with titers measured in immunogenicity assays.
Virosomal delivery of a short, conformationally constrained peptide derived from P. falciparum CSP induced a long-lived parasite-inhibitory antibody response in humans. Combination with a second virosomally-formulated peptide derived from P. falciparum AMA-1 did not interfere with the immunogenicity of either peptide, demonstrating the potential of influenza virosomes as a versatile, human-compatible antigen delivery platform for the development of multivalent subunit vaccines.
PLoS ONE 02/2007; 2(12):e1278. DOI:10.1371/journal.pone.0001278 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Plasmodium falciparum merozoite surface protein-1 19 kDa fragment (MSP-1(19)) comprises two closely packed EGF-like domains (EGF=epidermal growth factor), each stabilized by three disulfide bonds. The native conformation of this protein is important for eliciting P. falciparum growth inhibitory antibodies. Here we show that the N-terminal EGF domain alone can be chemically synthesized and efficiently refolded to a native-like state, as shown by its solution structure as determined by NMR spectroscopy. In order to study its immunogenicity, the domain was coupled through its N terminus to a phospholipid and incorporated into reconstituted influenza virus-like particles (virosomes). When used to immunize mice, the peptide-loaded virosomes elicited potent humoral immune responses that were shown by Western blots and immunofluorescence assays to cross-react with native MSP-1 on the surfaces of P. falciparum blood stage parasites. This opens the way for a medicinal chemistry-oriented approach to the study and optimization of the antigenicity of the protein as a potential malaria vaccine candidate, whilst exploiting the immunopotentiating properties of influenza virosomes as a delivery vehicle.
[Show abstract][Hide abstract] ABSTRACT: Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and invade hepatocytes as a first and obligatory step of the parasite life cycle in man. Hepatocyte invasion involves proteins secreted from parasite vesicles called micronemes, the most characterized being the thrombospondin-related adhesive protein (TRAP). Here we investigated the expression and function of another microneme protein recently identified in Plasmodium falciparum sporozoites, apical membrane antigen 1 (AMA-1). P. falciparum AMA-1 is expressed in sporozoites and is lost after invasion of hepatocytes, and anti-AMA-1 antibodies inhibit sporozoite invasion, suggesting that the protein is involved during invasion of hepatocytes. As observed with TRAP, AMA-1 is initially mostly sequestered within the sporozoite. Upon microneme exocytosis, AMA-1 and TRAP relocate to the sporozoite surface, where they are proteolytically cleaved, resulting in the shedding of soluble fragments. A subset of serine protease inhibitors blocks the processing and shedding of both AMA-1 and TRAP and inhibits sporozoite infectivity, suggesting that interfering with sporozoite proteolytic processing may constitute a valuable strategy to prevent hepatocyte infection.
[Show abstract][Hide abstract] ABSTRACT: Apical membrane antigen 1 (AMA-1) of Plasmodium falciparum is a leading candidate antigen for inclusion in a malaria subunit vaccine. Its ectodomain can be divided into three subdomains, each with disulfide bond-stabilized structures. Since the majority of antibodies raised against the ectodomain appear to recognize strain-specific epitopes in domain I, we attempted to develop a vaccine formulation which directs the immune response to a region that contains more conserved epitopes. Here we demonstrate that a virosomal formulation of a peptide that mimics the semiconserved loop I of domain III elicits parasite growth-inhibitory antibodies. A synthetic peptide comprising residues 446 to 490 of AMA-1 (AMA-1(446-490)) was conjugated through the N terminus to a derivative of phosphatidylethanolamine and the phosphatidylethanolamine-peptide conjugate was incorporated into immunopotentiating reconstituted influenza virosomes as a human-compatible antigen delivery system. Both cyclized and linear versions of the peptide antigen elicited antibodies which specifically bound to parasite-expressed AMA-1 in Western blotting with parasite lysates as well as in immunofluorescence assays with blood stage parasites. All 11 peptidomimetic-specific monoclonal antibodies generated were cross-reactive with parasite-expressed AMA-1. Antigen binding assays with a library of overlapping cyclic peptides covering the target sequence revealed differences in the fine specificity of these monoclonal antibodies and provided evidence that at least some of them recognized discontinuous epitopes. The two immunodominant epitopes comprised the conserved linear sequences K(459)RIKLN(464) and D(467)DEGNKKII(475). A key feature of the synthetic vaccine formulation proposed here is the display of the peptide antigen in a native-like state on the surface of the virosome.
Infection and Immunity 09/2003; 71(8):4749-58. DOI:10.1128/IAI.71.8.4749-4758.2003 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have identified a novel conserved protein of Plasmodium falciparum, designated D13, that is stage-specifically expressed in asexual blood stages of the parasite. The predicted open reading frame (ORF) D13 contains 863 amino acids with a calculated molecular mass of 99.7 kDa and displays a repeat region composed of pentapeptide motives. Northern blot analysis with lysates of synchronized blood stage parasites showed that D13 is highly expressed at the mRNA level during schizogony. The first N'-terminal 138 amino acids of D13 were expressed in Escherichia coli and the purified protein was used to generate anti-D13 monoclonal antibodies (MAbs). Using total lysates of blood stage parasites and Western blot analysis, these MAbs stained one single band of approximately 100 kDa, corresponding to the predicted molecular mass of ORF D13. Western blot analysis demonstrated further that D13 is expressed during schizogony, declines rapidly in early ring stages and is undetectable in trophozoites. D13 protein is localized in individual merozoites in a distinct area, as demonstrated by indirect immunofluorescence analysis. After subcellular fractionation, D13 was confined to the pelleted fraction of the parasite lysate and its extraction by alkaline carbonate buffer treatment indicated that D13 is not a membrane-integral protein. Inclusion of certain anti-D13 MAbs into in vitro cultures of blood stage parasites resulted in considerable reduction in parasite growth. The N'-terminal domain encompassing 158 amino acids is 94 and 95%, respectively, identical at the amino acid level between Plasmodium knowlesi, Plasmodium yoelii, and P. falciparum. In summary, we describe a novel stage-specifically expressed, highly conserved gene product of P. falciparum that is recognized by parasite growth inhibitory antibodies.
Infection and Immunity 05/2003; 71(4):2173-81. DOI:10.1128/IAI.71.4.2173-2181.2003 · 3.73 Impact Factor