Use of a recombinant baculovirus product to measure naturally-acquired human antibodies to disulphide-constrained epitopes on the P. falciparum merozoite surface protein-1 (MSP1)

Abstract An enzyme-linked immunosorbent assay (ELISA) has been developed to measure antibody levels in human sera to a candidate vaccine antigen, merozoite surface protein-1 (MSP1), of the malaria parasite Plasmodium falciparum . To ensure the detection of antibodies reactive with important conformational epitopes, antigens used in the ELISA were obtained from either in vitro parasite cultures, or from a baculovirus expression system in which correct folding of recombinant MSP1-derived polypeptides has been previously demonstrated. The specificity of the ELISA was confirmed using a novel antibody affinity select method. The assay was used to investigate the pattern of acquisition of anti-MSP1 antibodies in a cross-sectional survey of 387 3–8 year old residents of a malaria endemic area of the Gambia. A significant positive correlation between anti-MSP1 antibody levels and age was evident, though individual responses to two antigens corresponding to two distinct domains of the MSP1 varied widely.     

Protection against malaria induced by chirally modified Plasmodium falciparum's MSP-1 42 pseudopeptides

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.     

Association between sequence polymorphism in an epitope of Babesia divergens Bd37 exoantigen and protection induced by passive transfer

In Europe, Babesia divergens is the major agent responsible for babesiosis in cattle and can occasionally infect splenectomised humans. Recently, we reported the characterisation of a 37 kDa exoantigen (Bd37) anchored in the merozoite membrane of B. divergens by a glycosylphosphatidyl-inositol. After phospholipase hydrolyse of the glycosylphosphatidyl-inositol anchor, the Bd37 antigen could be isolated in the plasma of the infected host and from the in vitro culture supernatants. Immunisation of mice with a gel-filtration protective fraction of B. divergens exoantigens, produced a monoclonal antibody (MAb), called F4.2F8-INT, directed against Bd37. In the present study, we report data on passive protection using MAb F4.2F8-INT. This MAb was able to completely protect against virulent challenges with B. divergens isolates Rouen 1987 (Rouen87) and Weybridge 8843 (W8843) but had no protective effect against another French isolate from Massif Central (6303E). Physical characterisation of the epitope recognised by F4.2F8-INT allowed us to explain the differences observed between these isolates by western blotting and passive protection. These results suggest that the antigen carrying this epitope could be used as a target in the development of a recombinant vaccine against B. divergens babesiosis.     

Plasmodium vivax: Comparison of the immune responses between oral and parenteral immunization of rPv54 in BALB/c mice

The merozoite surface protein-1 (MSP-1) from Plasmodium vivax was evaluated as an oral vaccine candidate by cloning and expressing the interspecies conserved block 10 (ICB10) of the MSP-1 from a Korean isolate in Escherichia coli. The expressed fusion protein contained ICB10 and a maltose-binding protein (MBP), rPv54, has a molecular weight of approximately 54 kDa as determined by SDS-PAGE analysis. IgG against rPv54 was successfully produced in BALB/c mice by oral immunization and sustained for more than 4 months. IgG2b was dominantly produced in both oral and parenteral immunizations. The rPv54 increased the frequency of NK, NKT, CD4+ T, CD8+ T, and B cells in both immunizations. IL-5 and TNF-α were increased in both significantly. In conclusion, rPv54 might be a valuable potential vaccine candidate for the oral and parenteral immunization against vivax malaria.     

Trials for the co-expression of the merozoite surface protein-1 and circumsporozoite protein genes of Plasmodium vivax

Merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen, and circumsporozoite protein (CSP), a component of sporozoites that includes a Plasmodium vivax B-cell epitope, are strong candidates for use in a malaria vaccine. A chimeric recombinant gene containing portions of both msp-1 and csp from P. vivax separated by Pro-Gly linker motif was generated. The construct gene was named mlc (msp-1, linker, and csp). The MLC chimeric recombinant protein had a molecular weight of approximately 25 kDa when expressed in Escherichia coli, as determined with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis. The purified chimeric protein reacted with the sera of patients infected with P. vivax but not with the sera of uninfected patients according to western blot analysis. The chimeric protein reacted well with sera of malaria patients (109/115, 94.78%) as assessed with enzyme-linked immunosorbent assay (ELISA). BALB/c mice that were orally immunized with the MLC chimeric recombinant protein successfully produced antigen-specific antibodies. Additionally, levels of the Th1-associated cytokines IL-12(p40), TNF-α, and IFN-γ were significantly increased in the spleens of the BALB/c mice. Therefore, the E. coli-expressed MLC chimeric recombinant protein might be used as a valuable vaccine candidate for oral immunization against vivax malaria.     

Sequence diversity and molecular evolution of the merozoite surface antigen 2 of plasmodium falciparum

Eleven new alleles of the Plasmodium falciparum merozoite surface antigen 2 (MSA2) from Papua New Guinea were analyzed by direct sequencing of polymerase chain reaction (PCR) products. We have used the sequence information to trace the molecular evolution of MSA2. The repeats of ten alleles belonging to the 3D7 allelic family differed considerably in size, nucleotide sequence, and repeat copy number. In the repeat region of these new alleles, codon usage was extremely biased with an exclusive use of NNT codons. Another new allele sequenced belonged to the FC27 family and confirmed the family-specific conserved structure of 96 and 36 bp repeats. In order to assess sequence microheterogeneity within samples defined as the same genotype by restriction fragment length polymorphism (RFLP), we have analyzed single-strand conformation polymorphism (SSCP) of different samples of the most frequent allele (D10 of the FC27 family) in the study population. No sequence heterogeneity could be detected within the repeat region. Based on analysis of the repeat regions in both allelic families, we discuss the hypothesis of a different evolutionary strategy being represented by each of the allelic families. Received: 8 February 1995 / Accepted: 24 March 1997     

Plasmodium lophurae: the ultrastructure of the exoerythrocytic stages

The fine structure of the exoerythrocytic stages of Plasmodium lophurae was studied. in specimens grown in tissue cultures of avian cells. Specimens were prepared for sectioning by a method which minimizes disturbance and permits precise selection and orientation specimens.Plasmodium lophurae is similar in many aspects to P. fallax. Merozoites are highly specialized and differentiated. Analysis of their ultrastructure revealed the polar complex to be a specialization of the pellicular envelope and its associated underlying microtubules. The polar rings may simply be a modification of the inner membrane of the pellicle and not discrete structures as previously reported. The electron-dense polar organelles are separated on morphological grounds into three groups: the large paired organelles and the small dense bodies which are both linked to microducts, and the transitional bodies, a third organelle being reported for the first time. Transitional bodies are without microducts, occur in fully mature merozoites and persist only for a short period. All three of these organelles appear to be related to and possibly even derived from internal membrane systems and ribosomes. The apolar end of the merozoite contains the mitochondrion and its associated spherical body. Detailed study of the latter shows it to be cylindrical.Upon entering the host cell, the parasite adds a third membrane at the interface between it and the cell. The merozoite becomes spherical and undergoes transformation into a trophozoite. During this reorganization phase, dedifferentiation occurs and is followed by a rapid growth phase. The end of the growth phase is signaled by the appearance of germinal clefts and nuclear division. The entire process of schizogony culminates in a highly synchronized formation of merozoites.Processes of the limiting membrane forming the host parasite interface were observed extending deply into the cytoplasm of the host cell and often appeared to form bridges between two or more parasites. The significance of this new observation is not yet established.     

Construction, screening and identification of a phage display antibody library against the Eimeria acervulina merozoite

A single-chain antibody library against Eimeria acervulina merozoites was constructed by phage display approach. Antibody-displaying phage was selected in four panning rounds against cryopreserved E. acervulina merozoites. Five clones were randomly selected from the fourth panning round, and their nucleotide sequences were aligned and compared to mouse germ-line sequences. Soluble antibody was produced in a non-suppressor Escherichia coli strain, purified by protein A affinity chromatography, and characterized by Western-blotting. Immunofluorescence assay showed localization of the produced recombinant antibody fragment on the surface E. acervulina merozoites. These resultant antibody fragments showed high specificity and binding capacity for soluble antigens and intact fixed merozoites which seems promising as diagnostic, therapeutic and/or vaccine tools against coccidiosis.     

Identifying and characterising the Plasmodium falciparum merozoite surface protein 10 Plasmodium vivax homologue

Plasmodium vivax malaria is one of the most prevalent parasitic diseases in Asia and Latin-America. The difficulty of maintaining this parasite culture in vitro has hampered identifying and characterising proteins implied in merozoite invasion of red blood cells. We have been able to identify an open reading frame in P. vivax encoding the Plasmodium falciparum merozoite surface protein 10 homologous protein using the partial sequences from this parasite's genome reported during 2004. This new protein contains 479 amino-acids, two epidermal growth factor-like domains, hydrophobic regions at the N- and C-termini, being compatible with a signal peptide and a glycosylphosphatidylinositol anchor site, respectively. The protein is expressed during the parasite's asexual stage and is recognised by polyclonal sera in parasite lysate using Western blot. P. vivax-infected patients' sera highly recognised recombinant protein by ELISA.