53 research outputs found
Characterization of protective epitopes in a highly conserved Plasmodium falciparum antigenic protein containing repeats of acidic and basic residues
The delineation of putatively protective and immunogenic epitopes in vaccine candidate proteins constitutes a major research effort towards the development of an effective malaria vaccine. By virtue of its role in the formation of the immune clusters of merozoites, its location on the surface of merozoites, and its highly conserved nature both at the nucleotide sequence level and the amino acid sequence level, the antigen which contains repeats of acidic and basic residues (ABRA) of the human malaria parasite Plasmodium falciparum represents such an antigen. Based upon the predicted amino acid sequence of ABRA, we synthesized eight peptides, with six of these (AB-1 to AB-6) ranging from 12 to 18 residues covering the most hydrophilic regions of the protein, and two more peptides (AB-7 and AB-8) representing its repetitive sequences. We found that all eight constructs bound an appreciable amount of antibody in sera from a large proportion of P. falciparum malaria patients; two of these peptides (AB-1 and AB-3) also elicited a strong proliferation response in peripheral blood mononuclear cells from all 11 human subjects recovering from malaria. When used as carrier-free immunogens, six peptides induced a strong, boostable, immunoglobulin G-type antibody response in rabbits, indicating the presence of both B-cell determinants and T-helper-cell epitopes in these six constructs. These antibodies specifically cross-reacted with the parasite protein(s) in an immunoblot and in an immunofluorescence assay. In another immunoblot, rabbit antipeptide sera also recognized recombinant fragments of ABRA expressed in bacteria. More significantly, rabbit antibodies against two constructs (AB-1 and AB-5) inhibited the merozoite reinvasion of human erythrocytes in vitro up to ~90%. These results favor further studies so as to determine possible inclusion of these two constructs in a multicomponent subunit vaccine against asexual blood stages of P. falciparum
A novel Plasmodium falciparum rhoptry associated adhesin mediates erythrocyte invasion through the sialic-acid dependent pathway
Erythrocyte invasion by Plasmodium falciparum merozoites is
central to blood-stage infection and malaria pathogenesis. This
intricate process is coordinated by multiple parasite adhesins
that bind erythrocyte receptors and mediate invasion through
several alternate pathways. P. falciparum expresses 2700 genes
during the blood-stages, of which the identity and function of
many remains unknown. Here, we have identified and characterized
a novel P. falciparum rhoptry associated adhesin (PfRA) that
mediates erythrocyte invasion through the sialic-acid dependent
pathway. PfRA appears to play a significant functional role as
it is conserved across different Plasmodium species. It is
localized in the rhoptries and further translocated to the
merozoite surface. Both native and recombinant PfRA specifically
bound erythrocytes in a sialic-acid dependent, chymotrypsin and
trypsin resistant manner, which was abrogated by PfRA antibodies
confirming a role in erythrocyte invasion. PfRA antibodies
inhibited erythrocyte invasion and in combination with
antibodies against other parasite ligands produced an additive
inhibitory effect, thus validating its important role in
erythrocyte invasion. We have thus identified a novel P.
falciparum adhesin that binds with a sialic acid containing
erythrocyte receptor. Our observations substantiate the strategy
to block P. falciparum erythrocyte invasion by simultaneously
targeting multiple conserved merozoite antigens involved in
alternate invasion pathways
Identifying Immune Correlates of Protection Against Plasmodium falciparum Through a Novel Approach to Account for Heterogeneity in Malaria Exposure
Background: A main criterion to identify malaria vaccine
candidates is the proof that acquired immunity against them is
associated with protection from disease. The age of the studied
individuals, heterogeneous malaria exposure, and assumption of
the maintenance of a baseline immune response can confound these
associations. Methods: Immunoglobulin G/immunoglobulin M (IgG/
IgM) levels were measured by Luminex(R) in Mozambican children
monitored for clinical malaria from birth until 3 years of age,
together with functional antibodies. Studied candidates were
pre-erythrocytic and erythrocytic antigens, including
EBAs/PfRhs, MSPs, DBLs, and novel antigens merely or not
previously studied in malaria-exposed populations. Cox
regression models were estimated at 9 and 24 months of age,
accounting for heterogeneous malaria exposure or limiting
follow-up according to the antibody's decay. Results:
Associations of antibody responses with higher clinical malaria
risk were avoided when accounting for heterogeneous malaria
exposure or when limiting the follow-up time in the analyses.
Associations with reduced risk of clinical malaria were found
only at 24 months old, but not younger children, for IgG breadth
and levels of IgG targeting EBA140III-V, CyRPA, DBL5epsilon and
DBL3x, together with C1q-fixation activity by antibodies
targeting MSP119. Conclusions: Malaria protection correlates
were identified, only in children aged 24 months old when
accounting for heterogeneous malaria exposure. These results
highlight the relevance of considering age and malaria exposure,
as well as the importance of not assuming the maintenance of a
baseline immune response throughout the follow-up. Results may
be misleading if these factors are not considered
Falcipain-1, a Plasmodium falciparum Cysteine Protease with Vaccine Potential
Cysteine proteases (falcipains) of Plasmodium falciparum are potential targets for antimalarial chemotherapy, since they have been shown to be involved in important cellular functions such as hemoglobin degradation and invasion/rupture of red blood cells during parasite life cycle. The role of falcipain-1 at the asexual blood stages of the parasite still remains uncertain. This is mainly due to a lack of methods to prepare this protein in an active form. In order to obtain biologically active falcipain-1, a number of falcipain-1 constructs were designed and a systematic assessment of the refolding conditions was done. We describe here the expression, purification, and characterization of a falcipain-1 construct encoding mature falcipain-1 and 35 amino acids from the C-terminal of the pro domain. Recombinant falcipain-1 was overexpressed in the form of inclusion bodies, solubilized, and purified by Ni(2+)-nitrilotriacetic acid affinity chromatography under denaturing conditions. A systemic approach was then followed to optimize refolding parameters. An optimum refolding condition was obtained, and the yield of the purified refolded falcipain-1 was ∼1 mg/liter. Activity of the protein was analyzed by fluorometric and gelatin degradation assays. Immunolocalization studies using anti-falcipain-1 sera revealed a distinct staining at the apical end of the P. falciparum merozoites. Previous studies using falcipain-1-specific inhibitors have suggested a role of falcipain-1 in merozoite invasion. Based on its localization and its role in invasion, we analyzed the immunogenicity of falcipain-1 in mice, followed by heterologous challenge with Plasmodium yoelii sporozoites. Our results suggest a possible role of falcipain-1 in merozoite invasion
Antigenicity of a Bacterially Expressed Triple Chimeric Antigen of Plasmodium falciparum AARP, MSP-311 and MSP-119: PfAMSP-Fu35.
Development of fusion chimeras as potential vaccine candidates is considered as an attractive strategy to generate effective immune responses to more than one antigen using a single construct. Here, we described the design, production, purification and antigenicity of a fusion chimera (PfAMSP-Fu35), comprised of immunologically relevant regions of three vaccine target malaria antigens, PfAARP, PfMSP-3 and PfMSP-1. The recombinant PfAMSP-Fu35 is expressed as a soluble protein and purified to homogeneity with ease at a yield of ~ 7 mg L-1. Conformational integrity of the C-terminal fragment of PfMSP-1, PfMSP-119 was retained in the fusion chimera as shown by ELISA with conformation sensitive monoclonal antibodies. High titre antibodies were raised to the fusion protein and to all the three individual components in mice and rabbits upon immunization with fusion chimera in two different adjuvant formulations. The sera against PfAMSP-Fu35 recognized native parasite proteins corresponding to the three components of the fusion chimera. As shown by invasion inhibition assay and antibody mediated cellular inhibition assay, antibodies purified from the PfAMSP-Fu35 immunized serum successfully and efficiently inhibited parasite invasion in P. falciparum 3D7 in vitro both directly and in monocyte dependent manner. However, the invasion inhibitory activity of anti-AMSP-Fu35 antibody is not significantly enhanced as expected as compared to a previously described two component fusion chimera, MSP-Fu24. Therefore, it may not be of much merit to consider AMSP-Fu35 as a vaccine candidate for preclinical development
Co-dominant and reciprocal T-helper cell activity of epitopic sequences and formation of junctional B-cell determinants in synthetic T: B chimeric immunogens
The identification of defined T-helper (Th) cell determinants, particularly those recognized in the context of several MHC or HLA haplotypes, and their use to provide effective carrier help to short synthetic constructs representing a B-cell epitope have made it feasible to synthesize putatively potent immunogens. However, a number of crucial questions regarding immunogenicity of epitopic sequences need to be addressed before an optimally effective synthetic vaccine can be designed. The present study deals with the hybrid constructs incorporating a known B-cell epitope from the merozoite surface protein-1 (MSP-1) of a human malarial parasite, Plasmodium falciparum, and the promiscuous Th-cell epitope from tetanus toxin or from the circumsporozoite protein of P. falciparum. Here, we provide data which suggest that B- and T-cell determinants present in a hybrid construct could, in fact, provide reciprocal helper activity for antibody production; that antibodies to a Th-cell epitope may not necessarily block its helper function; and that functional B-cell epitopes may be formed. All this may influence, in an unpredictable manner, the quality of protective immune response sought to be generated using the chimeric immunogens, with important implications for vaccine design
Cloning and sequence analysis of the thrombospondin-related adhesive protein (TRAP) gene of Plasmodium cynomolgi bastianelli
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