[Show abstract][Hide abstract] ABSTRACT: Plasmodium vivax malaria remains one of the tropical diseases causing an enormous burden on global public health. Several proteins located on this parasite species' merozoite surface have been considered the most suitable antigens for being included in an anti-malarial vaccine, given the functional role they play during the parasite's interaction with red blood cells. The present study identifies and characterizes the P. vivax Pv12 surface protein which was evaluated by using molecular biology and immunochemistry assays; its antigenic potential was also examined in natural and experimental P. vivax malaria infections. The P. vivax VCG-1 strain Pv12 gene encodes a 362 amino acid-long protein exhibiting a signal peptide, a glycosylphosphatidylinositol (GPI) anchor sequence and two 6-Cys domains. The presence of the Pv12 protein on the parasite's surface and its association with detergent-resistant membrane complexes, together with its antigenic potential, supports the notion that this antigen could play an important role as a red blood cell binding ligand. Further studies aimed at establishing the immunogenicity and protection-inducing ability of the Pv12 protein or its products in the Aotus experimental model are thus suggested.
[Show abstract][Hide abstract] ABSTRACT: Effector mechanisms responsible for providing protective immunity against Plasmodium vivax (Pv) infection were examined in Aotus monkeys vaccinated with two Pv Merozoite Surface Protein-1 (PvMSP-1) recombinant polypeptides that had previously been shown to protect vaccines against parasite challenge. Vaccine efficacy was reproducible in this trial, showing that one out of the five monkeys immunised with the recombinant protein mixture was partially protected while three others controlled parasitaemia. Antibodies reactive to the parasite's native proteins, the recombinant polypeptides and peptides spanning both recombinant fragments were detected in most vaccinees. Despite substantial Peripheral Blood Mononuclear Cell (PBMC) antigen-specific cellular proliferation not being detected, high rPvMSP-1(20) specific gamma interferon (IFN-gamma) production was found in the three animals that controlled parasitaemia. Altogether the results suggest that antibody titres and antigen-specific IFN-gamma production mediate protective immunity against P. vivax.
[Show abstract][Hide abstract] ABSTRACT: The search for a rational method of developing an antimalarial vaccine (malaria caused by Plasmodium falciparum) consists of blocking receptor-ligand interaction. Conserved peptides derived from proteins involved in invasion and having strong red blood cell binding ability have thus been identified; immunization studies using Aotus monkeys revealed that these peptides were neither immunogenic nor protection-inducing. Some of these peptides induced long-lasting and very high antibody titers and protection when their critical red blood cell binding residues were replaced to change their immunological properties. Others induced short-lived antibodies that were not associated with inducing protection. The three-dimensional structure of the short-lived antibody-inducing peptide was determined by (1)H NMR. Their HLA-DRbeta1* molecule binding ability was also determined to ascertain the relationship among three-dimensional structure, their ability to bind to major histocompatibility complex class II molecules (MHC II), and possible short-lived antibody production. These short-lived antibody-inducing peptides were 6.8 +/- 0.5 A shorter between those residues theoretically coming into contact with pocket 1 and pocket 9 of HLA-DRbeta1* molecules to which they bind than immunogenic and protection-inducing peptides. These more compact alpha-helical structures suggest that these short-lived antibody-inducing peptides could have a structure more similar to those of native peptides than immunogenic and protective ones. Such shortening was associated with a shift in HLA-DRbeta1* molecule binding and a consequent shift in functional register reading, mainly by alleles of the same haplotype when compared with immunogenic protection-inducing HABPs, suggesting an imperfect and different conformation of the MHC II peptide-TCR complex.
[Show abstract][Hide abstract] ABSTRACT: The C-terminal portion of the Plasmodium falciparum blood stage MSP-1 antigen plays a key role in invasion of human erythrocytes. The MSP-1(1282-1301) non-polymorphic 1585 peptide, from the processed MSP-1(42) fragment, is poorly immunogenic and highly alpha-helical [Angew. Chem. Int. Ed. 40 (2001) 4654]. Assessing the alpha-carbon asymmetry and its implication in the host immune response is proposed in this work to overcome the 1585 peptide's immunological properties. Accordingly, the effect of incorporating single D-amino acids and psi-[CH(2)-NH] isoster bonds into the 1585 peptide was examined both at the immunogenic and 3D-structure levels. Therefore, specific binding to RBCs is promoted by site-directed chiral modifications on the native peptide as well as by simultaneously combining specific D-substitutions with psi-[CH(2)-NH] isoster bonds transforming this molecule into a high specific HLAbeta1*1101 allele binder. D-analog pseudopeptide immunized animals induced antibodies selectively recognizing a recombinant as well as native MSP-1(42) and MSP-1(33) fragments. Protection and low parasitemia levels were induced in Aotus monkeys immunized with the EVLYL(dK)PLAGVYRSLKKQLE analog. Peptide alpha-carbon chiral transformation is therefore an important target for structural modulation and, consequently, represents a novel approach towards designing multi-component subunit-based malarial vaccines.
Biochemical and Biophysical Research Communications 05/2005; 329(3):1053-66. DOI:10.1016/j.bbrc.2005.01.165 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two E. coli expressed recombinant polypeptides (rPvMSP-1(14) and rPvMSP-1(20)) contained in the 33kDa fragment, located within Plasmodium vivax merozoite surface protein (PvMSP-1) 42kDa C-terminal region, and a cocktail of high reticulocyte binding synthetic peptides located within these fragments, were evaluated for immunogenicity and protective immune responses in splenectomised and spleen intact Aotus nancymaae monkeys. Thirty splenectomised monkeys who had been previously immunised with either rPvMSP-1(14), rPvMSP-1(20), or a mixture of both recombinant fragments were intravenously challenged with the heterologous P. vivax VCG-1 strain (as determined by DNA sequencing); full protection was observed in five monkeys and low parasitaemia levels were obtained in eight more monkeys. Splenectomised control monkey group rapidly developed high parasitaemia levels, while no significant parasitaemia was obtained in the non-splenectomised control group. Although PvMSP-1 42 and 33kDa fragments were recognised by Western Blot and whole parasites by IFAT when tested with immune monkey sera, no correlation between protection and antibody titres by IFAT and ELISA was observed, suggesting that protection is not being solely mediated by a humoral immune response. This data showed that partial protection against a heterologous strain challenge was best achieved when immunising with a rPvMSP-1(14)-rPvMSP-1(20) mixture (2 were fully protected and 4 with low parasitaemia out of 12) suggesting for the first time, that these fragments could be good candidates for inclusion in a P. vivax multi-stage, multi-antigen vaccine.
[Show abstract][Hide abstract] ABSTRACT: The merozoite surface protein-1 represents a prime candidate for development of a malaria vaccine. Merozoite surface protein-1 has been shown to demonstrate high-activity peptide binding to human red blood cells. One of the high-activity binding peptides, named 5501, located in the N-terminus (amino acid sequence MLNISQHQCVKKQCPQNS) of the 19-kDa molecular mass fragment of merozoite surface protein-1, is conserved, nonimmunogenic and nonprotective. Its critical binding residues were identified and replaced with amino acids of similar mass but different charge, in order to modify their immunogenic and protective characteristics. Three analogues with positive or negative immunological results were studied by nuclear magnetic resonance to correlate their three-dimensional structure with their biological functions. The studied peptides presented alpha-helical fragments, but in different peptide regions and extensions, except for randomly structured 5501. We show that altering a few amino acids induced immunogenicity and protectivity against experimental malaria and changed the peptide three-dimensional structure, suggesting a better fit with immune-system molecules.
European Journal of Biochemistry 11/2003; 270(19):3946-52. DOI:10.1046/j.1432-1033.2003.03780.x · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Merozoite Surface Protein-1 (MSP-1) has been considered as a malaria vaccine candidate. It is processed during the Plasmodium falciparum invasion process of red blood cells (RBCs). A conserved MSP-1 C-terminal peptide was identified as a high-activity erythrocyte-binding peptide (HAEBP) termed 1585. Since conserved HAEBPs are neither antigenic nor immunogenic we decided to assess the significance of a single peptide bond replacement in 1585. Thus, two pseudopeptides were obtained by introducing a Y[CH2-NH] reduced amide isoster into the 1585 critical binding motif. The pseudopeptides bound to different HLA-DR alleles, suggesting that backbone modifications affect MHC-II binding patterns. Pseudopeptide-antibodies inhibit in vitro parasite RBC invasion by recognizing MSP-1. Each pseudopeptide-induced antibody shows distinct recognition patterns. 1H-NMR studies demonstrated that isoster bonds modulate the pseudopeptides' structure and thus their immunological properties, therefore representing a possible subunit malaria vaccine component.
[Show abstract][Hide abstract] ABSTRACT: Rational design of a malaria vaccine may result from the structure-immunogenicity relationship of analogues of peptide 1585, a protein sequence located at the N-terminal end of the P. falciparum 42-kDa merozoite surface protein-1. Altering a few amino acids in peptide 1585 (left) induces immunogenicity and protectivity, and changes the 3D structure, as shown for one of the analogues synthesized (right).
[Show abstract][Hide abstract] ABSTRACT: A double-blind randomized placebo-controlled field trial with the SPf66 malaria vaccine was carried out in an endemic area consisting of 14 small villages with exclusive fluvial access, in a rain forest area along the Rosario River, Colombia. A total of 1257 subjects completed the full three dose vaccination schedule on days 0, 30 and 180 (643 vaccinated group/623 placebo group) and were followed-up by passive and active surveillance over a period of 22 months. One hundred and thirty-four Plasmodium falciparum malaria episodes were detected (53 in vaccinated group/81 in placebo group), yielding an attack rate of 5.47 cases/100 person years of follow-up (pyears) in the vaccine group and 8.44/100 pyears in the placebo group. The estimated vaccine protective efficacy was 35.2% (95% CI 8.4-54.2%, P = 0.01). This result supports earlier findings that the SPf66 malaria vaccine diminishes the risk of infection by P. falciparum in endemic areas of South America.
[Show abstract][Hide abstract] ABSTRACT: SPf66 is a chemically synthesized 45 amino acid peptide derived from fractions of four different proteins of Plasmodium falciparum (83, 55 and 35 kDa and CS, the circumsporozoite protein) that elicits a protective immune response against malaria. In this paper we show the characterization of the SPf(66)n in batch 9 to be used in a field trial in young children at Ifakara in Tanzania. The analysis of SPf(66)n indicates that it is highly soluble in water and that the amino acid composition and sequence corresponds to that designed for the synthesis of the polypeptide. The packed product has a molecular weight ranging from 10 to 25 kDa. It is pure, free of metallic contaminants, atoxic and stable at 4 degrees C. The antibodies raised against this product in rabbits recognize the individual antigenic determinants of the molecule and the native epitopes of merozoites.
[Show abstract][Hide abstract] ABSTRACT: As part of the first trial of the SPf66 malaria vaccine in Africa, three randomized double-blind placebo-controlled studies of SPf66 have been conducted in a highly endemic area of Tanzania. The objectives were to confirm that the product is immunogenic and safe in highly exposed individuals. Results from ten male adult expatriates indicated that the product used in Tanzania is at least as immunogenic as that used in Colombia. No major side-effects were observed in indigenous SPf66 recipients (18 adults, and 25 children aged 1-4 years). Anti-SPf66 antibody titres in all groups showed clear responses to three doses of the vaccine.
[Show abstract][Hide abstract] ABSTRACT: Two independent vaccination trials using a hybrid synthetic polypeptide containing epitopes from four proteins of Plasmodium falciparum were performed. In the first trial 63 and in the second 122 volunteers were vaccinated, using different immunization schedules. The analysis of the humoral response to the vaccine, measured by IgG antibody titres to the polypeptide showed a bimodal distribution in both cases suggesting genetic control of the immune response to this protein. There was a small group of low or non-responders and a large group of good responders. HLA phenotyping of the two groups disclosed an association of the low responders to HLA-DR4 antigens with chi-square P value of 0.00039 when compared with the good responders group. These findings provide evidence for the genetic control of the immune response to the synthetic vaccine by the association of this response with particular alleles of the HLA class II antigens; such findings may lead to an explanation of the mechanism involved in disease susceptibility and need to be used in the design of a totally effective vaccine.