Peptides for inducing cross-reactive anti-malarial antibodies.

Immunological cross-reactions between malarial proteins are frequently observed. This paper describes an approach to generate such antibody responses that target many parasite proteins using a limited number of peptides as immunogens. Peptides used contained a known epitope, NKND, that is common to many malarial proteins or combinations of tri-peptides which are commonly present in parasite proteins. One of the two NKND-containing peptides elicited antibodies reacting to six parasite proteins and fusion proteins containing NKND. The antibody specificity was directed to NKND. Two of the four combination peptides were recognized by hyperimmune human sera and mouse immune serum in vitro, and one elicited antibodies recognizing parasites on immunofluorescence assay.

[Ultrastructural localization of 145/102 kDa antigens in erythrocytic stages of Plasmodium falciparum].

The ultrastructural localization of the 145/102 kDa antigens recognized by the possible protective monoclonal antibody (McAb) M26-32 in erythrocytic stages of Plasmodium falciparum, FCC1/HN, in vitro, was investigated by immuno-electron microscopy with LR White resin embedding and colloidal gold probe cytochemistry techniques. The results showed that the gold particles were mainly localized within the cytoplasm of ring forms, trophozoites, schizonts and merozoites of the Plasmodium. Some gold particles were found to locate on the pellicular complex of the plasmodium surface or in the cytoplasm of the infected erythrocytes. The results indicated that 145/102 kDa antigens were the common cytoplasmic antigens of asexual blood stages of Plasmodium falciparum, FCC1/HN, while a portion of the antigens could be transported to the cytoplasm of the infected erythrocytes via the pellicular complex of the plasmodium surface (Figs. 1-4).

[Ultrastructural localization of 185 kDa and 82/41 kDa protective antigens in Plasmodium falciparum, FCC1/HN].

Plasmodium falciparum FCC1/HN-infected human erythrocytes were embedded with LR White resin at low temperature. The 185 kDa and 82/41 kDa proteins in erythrocytic stages of P. falciparum were then immunolabeled by using the protective monoclonal antibodies (McAb)F6-D3 and F6-C2 with protein A-colloidal gold probe. The electron-microscopical observation showed that the 185kDa protein recognized by McAb F6-D3 was located on the surface of free and intracellular merozoites as well as the cytoplasm, plasma membrane, and parasitophorous vacuole membrane of immature schizonts. The 82/41 kDa proteins identified by McAb F6-C2 was located within the rhoptries of immature schizonts and mature merozoites. These results demonstrated ultrastructurally that the 185 kDa and 82/41 kDa protective antigens were merozoite surface antigen and merozoite rhoptry antigens of P. falciparum FCC1/HN, respectively.

[Localization of the antigen in erythrocytic stages of Plasmodium yoelii by immuno-electron microscopy].

In this study, the antigen recognized by the protective McAb M26-32 in erythrocytic stages of P. yoelii was localized by immuno-electron microscopy with LR Whithe resin embedding and colloidal gold probe cytochemical techniques. The results indicated that the antigen which reacts specifically to McAb M26-32 was mainly localized within the cytoplasm of early and late trophozoites, schizonts and merozoites, being the common antigen of asexual blood stages of the plasmodium. The amount of the antigen was on the increase during the development of trophozoite, while a portion of the antigen might be transported outward by exocytosis of the parasites and then be localized in the cytoplasm of the infected erythrocytes adjacent to the parasites.

Identification of a common Plasmodium epitope (CPE) recognised by a pan-specific inhibitory monoclonal antibody.

A Plasmodium falciparum genomic expression library was screened with a monoclonal antibody produced from mice infected with Plasmodium yoelii. Eleven unique clones were isolated all of which contained the sequence NKND, IKND or KKND. This sequence was confirmed as the epitope of M26-32 by testing a series of overlapping peptides and the allowable substitutions determined by testing the binding of M26-32 to peptides containing all possible single amino acid replacements of NKND. Potential epitopes of M26-32 occur in many plasmodial proteins and this is consistent with the large number of proteins recognised in these parasites by Western blotting. Since this monoclonal antibody shows marked in vitro inhibition of P. falciparum growth, these data suggest that an anti-malarial vaccine may be produced by targeting such common plasmodial epitopes without necessarily identifying the corresponding antigens.

Studies on the mechanism of anemia in rodent malaria.

The underlying cause of anemia is one of the problems to be solved in malaria research. Many factors are involved in reducing the quantity of uninfected red blood cells (RBC) in addition to those infected RBC destroyed by malaria parasites. In the Plasmodium yoelii (P.y.)-mouse model, the amount of [51Cr]-labelled normal mouse RBC destroyed in peripheral blood as well as the quantity phagocytized by spleen cells during acute and chronic infection in vivo is reported in this paper. Our results show that compensatory enlargement of the spleen, which cleans up a large amount of the damaged uninfected RBC, may be the major cause of anemia in chronic malaria infection. In acute malaria infection destruction of uninfected RBC in peripheral circulation is higher than that in normal mice. Neither malaria antigen, mouse autoantibody nor immune complex was detected on the surface of normal RBC from infected mice using indirect immunofluorescence assay (IFA) or [3H]-isoleucine-labelled P.y. antigen (P.y.Ag) in vitro. This suggests that malaria immune complexes do not play an important part in RBC destruction in circulating blood. Since no obvious hemolysis was observed by mixing RBC with P.y. culture supernatant in vitro, it is possible that physical and chemical changes in uninfected RBC induced by malaria metabolites are the prerequisite for their destruction in circulating blood in vivo. Hemolysis occurs due to external stresses, such as those incurred when damaged RBC run into each other in the blood stream or when they change their shape to pass through capillaries.

Relationship between three different B cell types and the specific efficiency of hybridoma formation.

The kinetic changes of the number of antigen-specific B cell (ASC, sIg+, Ig-non-secreting), preplasma cell (sIg+, Ig-secreting) and plasma cell (sIg-, Ig-secreting) in the spleens of inbred sibling Balb/c mice during the continuous OA immunization process were assayed, and their relationship to the corresponding specific efficiency of the hybridoma formed was compared. The results show: the specific efficiency of hybridoma formation is parallel with the kinetic changes of the number of ASC, but not with the number of preplasma or plasma cells. So we suggest that the antigen activated B lymphocytes in proliferating state, but neither the antibody-secreting cells (i.e. preplasma cell and plasma cell) nor small B lymphocytes are partners fused with the myeloma cells to yield functional hybridoma cells.

Identification of anti-human IgG monoclonal antibodies and monoclonal antibodies with confined subclass reactivity.

In a previous paper, 4 hybridoma cell lines (1B3, 2D5, 2A4 and 2B6) secreting antibodies against heavy chain of human IgG were reported. In this paper a more detailed study of these McAbs was introduced. First of all they were studied with double immunodiffusion test in agarose and it was demonstrated that they were reactive against 3 distinct antigenic determinants of human IgG. This result was also confirmed by ELISA competition test. These McAbs were further analysed for human IgG subclass proteins and it was found that 1B3 McAb gave positive reaction with all subclasses of IgG while the other 3 possessed confined subclass reactivity, i.e. McAb 2A4 did not react with IgG3 (called non-IgG3 McAb), and both McAbs 2D5 and 2B6 did not react with IgG4 (called non-IgG4 McAb). By agarose immunodiffusion test it was shown that: (i) Only transparent precipitation line was formed when McAb reacted with IgG antigen, however, in agarose containing 2% PEG-6000, the transparent precipitation line becomes opaque; (ii) When a mixture of two McAbs against the same antigenic determinants (2D5 and 2B6) was placed into the same well, the precipitation line remained transparent, but if they were not identical (2A4 and 2D5), then opaque precipitation line formed even in the absence of PEG. It was suggested that this phenomenon should be used for preliminary analysis for the identity among McAbs. The confined IgG subclass McAb may be used for the preparation and purification of IgG3 or IgG4 protein before specific McAb against IgG3 or IgG4 is obtained.(ABSTRACT TRUNCATED AT 250 WORDS)