Search

An effective malaria vaccine is a public health priority. Proteins expressed during the blood-stage of the parasite life cycle have been proposed as good vaccine candidates. No such blood-stage vaccine, however, is available against Plasmodium falciparum, the deadliest Plasmodium species. We show here that P. falciparum serine repeat antigen 5 (SERA5) is a potential vaccine immunogen. We have constructed a new recombinant molecule of SERA5, namely SE36, based on previously reported SE47' molecule by removing the serine repeats. Epidemiological study in the holo-endemic population of Solomon Islands shows highly significant correlation of sero-conversion and malaria protective immunity against this antigen. Animal experiments using non-human primates, and a human phase 1a clinical trial assessed SE36 vaccine immunogenicity. Vaccination of squirrel monkeys with SE36 protein and aluminum hydroxyl gel (SE36/AHG) conferred protection against high parasitemia and boosted serum anti-SE36 IgG after P. falciparum parasite challenge. SE36/AHG was highly immunogenic in chimpanzees, where serum anti-SE36 IgG titers last more than one year. Phase 1a clinical trial (current controlled trials, ISRCTN78679862) demonstrated the safety and immunogenicity of SE36/AHG with 30 healthy adults and 10 placebo controls. Three subcutaneous administrations of 50 and 100microg dose of SE36/AHG were well-tolerated, with no severe adverse events; and resulted in 100% sero-conversion in both dose arms. The current research results for SE36/AHG provide initial clinical validation for future trials and suggest clues/strategies for further vaccine development.

To clarify evolution and phylogenetic relationships of trypanosome alternative oxidase (AOX) molecules, AOX genes (cDNAs) of the African trypanosomes, Trypanosoma congolense and Trypanosoma evansi, were cloned by PCR. Both AOXs possess conserved consensus motifs (-E-, -EXXH-). The putative amino acid sequence of the AOX of T. evansi was exactly the same as that of T. brucei. A protein phylogeny of trypanosome AOXs revealed that three genetically and pathogenically distinct strains of T. congolense are closely related to each other. When all known AOX sequences collected from current databases were analyzed, the common ancestor of these three Trypanosoma species shared a sister-group position to T. brucei/T. evansi. Monophyly of Trypanosoma spp. was clearly supported (100% bootstrap value) with Trypanosoma vivax placed at the most basal position of the Trypanosoma clade. Monophyly of other eukaryotic lineages, terrestrial plants + red algae, Metazoa, diatoms, Alveolata, oomycetes, green algae, and Fungi, was reconstructed in the best AOX tree obtained from maximum likelihood analysis, although some of these clades were not strongly supported. The terrestrial plants + red algae clade showed the closest affinity with an alpha-proteobacterium, Novosphingobium aromaticivorans, and the common ancestor of these lineages, was separated from other eukaryotes. Although the root of the AOX subtree was not clearly determined, subsequent phylogenetic analysis of the composite tree for AOX and plastid terminal oxidase (PTOX) demonstrated that PTOX and related cyanobacterial sequences are of a monophyletic origin and their common ancestor is linked to AOX sequences.

Calpain is a calcium-dependent cystein protease, and the homologues of schistosome are known as one of vaccine candidate molecules against schistosomiasis. Here, we established two IL-2 producing T cell hybridoma cell lines specific for Schistosoma japonicum calpain, to identify T cell epitope(s) on the molecule. Overlapping 15mer oligopeptides of calpain were synthesized and tested for their stimulatory abilities to the hybridomas. As a result, epitopes recognized by the two hybridoma lines were the same: EQLKIYAQRC. Spleen cells from calpain multiple antigenic peptide (MAP)-immunized BALB/c mice produced IFNgamma upon stimulation with MAP or soluble worm antigen preparation (SWAP). The identification of the T cell epitope to stimulate Th1 response will contribute to the proper design of synthetic vaccines, evaluation of their protective potentials and elucidation of protective mechanisms in murine experimental schistosomiasis.

A monoclonal antibody was generated against the large subunit of Schistosoma japonicum calpain to study the localization and possible function of the molecule in vivo. Mice were immunized with recombinant S. japonicum calpain and polyclonal antisera and a monoclonal antibody specific to schistosome calpain was obtained. In immunohistochemistry, a monoclonal antibody against S. japonicum calpain, KG-2E11, bound weakly to calpain expressed at the surface of adult worm tegument, however, it bound strongly to the cercarial secretions ("footprints") of S. japonicum, emitted from the penetration glands. The present study indicates that calpain is multifunctional as it is expressed at various locations in different developmental stages. Calpain-based vaccines could thus possibly induce protective immunity against cercariae and the following early developing stages.

Cryptosporidium parvum is a parasitic protozoan that causes the diarrheal disease cryptosporidiosis, for which no satisfactory chemotherapy is currently available. Although the presence of mitochondria in this parasite has been suggested, its respiratory system is poorly understood due to difficulties in performing biochemical analyses. In order to better understand the respiratory chain of C. parvum, we surveyed its genomic DNA database in GenBank and identified a partial sequence encoding cyanide-insensitive alternative oxidase (AOX). Based on this sequence, we cloned C. parvum AOX (CpAOX) cDNA from the phylum apicomplexa for the first time. The deduced amino acid sequence (335 a.a.) of CpAOX contains diiron coordination motifs (-E-, -EXXH-) that are conserved among AOXs. Phylogenetic analysis suggested that CpAOX is a mitochondrial-type AOX, possibly derived from mitochondrial endosymbiont gene transfer. The recombinant enzyme expressed in Escherichia coli showed quinol oxidase activity. This activity was insensitive to cyanide and highly sensitive to ascofuranone, a specific inhibitor of trypanosome AOX.

Background: MSP-1 of Plasmodium falciparum induces strong prohferative T cell responses even in malaria-nonexposed individuals. Epitopes recognized by malaria-nonimmune T cells have not been identified, and immunological mechanisms inducing such T cell responses remain to be uncovered. MSP-1 is a vaccine candidate, and it should be understood whether those epitopes have any roles in MSP-1-mediated protective immunity. The T epitopes-inducing malaria-naive T cell response was analyzed in the hope of understanding the underlying mechanisms. Methods: Human T cell lines and clones reactive to MSP-1 of P. falciparum were established from malaria-nonexposed Japanese donors in vitro, and epitope peptides were identified. Sequences of those epitope peptides were compared to unrelated peptides in the data base. One of those peptides was tested for both binding to HLA-DR molecules and inducing prohferative responses of MSP-1-reactive T cells. Results: There are at least 6 epitopes recognized by malaria-naive T cells under the restriction by HLA-DRBΓ1502 or 0802. Important amino acids for the T cell recognition were identified for an MSP-1 pep-tide. A yeast peptide which shared those residues induced prohferative responses of MSP-1-reactive T cells. Conclusion: We identified T epitopes in the N-terminal region of MSP-1, some of which showed molecular similarities with unrelated environmental antigens, suggesting the presence of cross-reactive T epitopes in MSP-1. Cytokine production in response to those epitopes suggests regulatory functions of those T cells during primary infection with P. falciparum.

Binding capacities of synthetic peptides to HLA-DR molecules were tested on filter papers to identify putative helper T-cell epitopes on a malarial protein. The antigen tested was the merozoite surface glycoprotein 1 (MSP1) of Plasmodium falciparum, a vaccine candidate targeting the asexual erythrocytic stage. Bindings between synthetic oligopeptides and HLA-DR molecules were tested. Such bindings were not non-specific, and a known helper T-cell epitope peptide showed positive binding to the restricting HLA-DR molecule. By using this screening system, we observed the unequal distribution of HLA-DR-binding peptides in 10 out of 17 MSP1 blocks tested. Block #6 of MSP1 seemed to show the highest frequency in the positive binding; on the other hand, blocks #1 and #17, both of which were thought to be vaccine candidate regions, contained fewer HLA-DR binding peptides. This was not inconsistent with the results that block #17 was less stimulatory to peripheral T cells than block #6. The peptides with positive binding to HLA-DR showed actual epitope activities when we tested peptide-driven proliferation of human bulk T-cell lines, and association between the two parameters was statistically significant (P

We analyzed the relationships between levels of antibody specific for merozoite surface glycoprotein-1 (MSP1) of Plasmodium falciparum and clinical manifestations in humans. We prepared recombinant MSP1 proteins representing block 3 (M3), block 6 (M6), blocks 1–6 (M1/6), and block 17. When we divided the slide-positive individuals in Guadalcanal into symptomatic and asymptomatic groups, the former group showed lower IgG levels against M6 and block 17, but not against M3, than did the asymptomatic group (P < 0.01). The possibility of nonspecific suppression was unlikely, given that the levels of antibody against poliomyelitis virus observed in the two groups were almost the same. Among the IgG subclasses tested, production of cytophilic IgG3 seemed to be dominant. When we analyzed epitopes recognized by antibodies against block 17, a peptide (SSSNFLGIS) was preferentially recognized by sera from asymptomatic individuals. These results suggest that clinical symptoms occurring during falciparum malaria seem to be associated with the development of levels of antibody against particular epitopes on MSP1, which is under the control of an immunoregulatory mechanism.