Enzyme-linked staining method for light microscopic detection of antibodies to parasite antigens on the membrane of Plasmodium falciparum infected erythrocytes.

Indirect erythrocyte membrane immunofluorescence (EMIF), a standard method for detection of antibodies to the P. falciparum blood stage vaccine candidate antigen Pf155/RESA, has been adapted to light microscopy by enzyme-linked immunostaining of the erythrocyte membrane, using alkaline phosphatase and chromogenic substrate. This method gives a dark blue staining of the membranes of ring infected erythrocytes. Results obtained with 70 African sera in EMIF and in enzyme immunostaining correlated well although the enzyme based method sometimes resulted in higher antibody titers and appeared to be slightly more sensitive. Similar results were obtained when comparing immunofluorescence with enzyme immunostaining for detection of antibodies to intraerythrocytic parasite antigens. The enzyme linked immunostaining described is simple and fast and does not require expensive equipment and should, thus, be well suited for use in laboratories with limited resources or under field conditions.

Epitope specificity and capacity to inhibit parasite growth in vitro of human antibodies to repeat sequences of the Plasmodium falciparum antigen Ag332.

It has earlier been shown that the Plasmodium falciparum-reactive human monoclonal antibody 33G2 inhibits parasite growth in vitro as well as cytoadherence of infected red blood cells to melanoma cells in vitro. MoAb 33G2 recognizes an epitope of the P. falciparum antigen Ag332 and cross-reactive determinants in Pf155/RESA and Pf11.1 located in repetitive regions containing sequences of regularly spaced pairs of glutamic acid. To study whether antibodies of this specificity frequently occur in human immune sera and if they could be of importance for protective immunity, antibodies were affinity purified on MoAb 33G2 reactive Ag332 peptides. The epitope specificity of the affinity purified antibodies, determined by the Pepscan method, resembled that of MoAb 33G2, but showed differences in fine specificity. The antibodies cross-reacted to some extent with Pf11.1 and Pf155/RESA repeat peptides as detected by peptide ELISA and Pepscan. In indirect immunofluorescence all purified antibodies displayed a dotted pattern of staining of late stage infected red blood cells of two lines of the P. falciparum strain FCR3, including a Pf155/RESA deficient line. The in vitro growth of these two lines was efficiently inhibited by the affinity purified antibodies, indicating that their inhibitory effect was mainly due to reactivity with antigens other than Pf155/RESA. This, and the fact that Pf11.1 has been shown not to be expressed by the asexual stages suggests that Ag332 may be an important target for potentially protective antibodies in vivo and that Ag332 based immunogens are of interest for development of malaria subunit vaccines.

Wild isolates of Plasmodium falciparum malaria show decreased sensitivity to in vitro inhibition of parasite growth mediated by autologous host antibodies.

Antigenic diversity in field populations of Plasmodium falciparum parasites may delay the acquisition of protective immunity to malaria, the development of which may thus require repeated exposure to infection over a prolonged period of time. In this study we show that P. falciparum parasites may vary in their sensitivity to antibody-mediated invasion/growth inhibition in vitro. Wild isolates of P. falciparum from children living in an endemic area of Burkina Faso were tested for their sensitivity to the growth inhibitory effects of antibodies originating from the same (autologous) and from other donors (heterologous). A significantly lower invasion inhibition activity was obtained when the isolates and antibodies were tested in autologous compared with heterologous combinations. The lower sensitivity to growth inhibition by autologous antibodies may be due to immune pressure in vivo, selecting from a heterogeneous parasite population those with a low expression of the antigens recognized by the host's antibodies. Alternatively, the parasites cultured from each child might represent expanding parasite populations, mainly constituting strains not earlier seen by the immune system of that specific host. The results reinforce the concern about Plasmodium antigenic diversity as a major obstacle towards the development of an effective malaria vaccine.

Immunoglobulin E, a pathogenic factor in Plasmodium falciparum malaria.

Most children and adults living in areas where the endemicity of Plasmodium falciparum malaria is high have significantly elevated levels of both total immunoglobulin E (IgE) and IgE antimalarial antibodies in blood. This elevation is highest in patients with cerebral malaria, suggesting a pathogenic role for this immunoglobulin isotype. In this study, we show that IgE elevation may also be seen in severe malaria without cerebral involvement and parallels an elevation of tumor necrosis factor alpha (TNF). IgE-containing serum from malaria immune donors was added to tissue culture plates coated with rabbit anti-human IgE antibodies or with P. falciparum antigen. IgE-anti-IgE complexes as well as antigen-binding IgE antibodies induced TNF release from peripheral blood mononuclear cells (PBMC). Nonmalaria control sera with no IgE elevation induced significantly less of this cytokine, and the TNF-inducing capacity of malaria sera was also strongly reduced by passing them over anti-IgE Sepharose columns. The cells giving rise to TNF were adherent PBMC. The release of this cytokine probably reflects cross-linking of their low-affinity receptors for IgE (CD23) by IgE-containing immune complexes known to give rise to monocyte activation via the NO transduction pathway. In line with this, adherent monocytic cells exposed to IgE complexes displayed increased expression of CD23. As the malaria sera contained IgG anti-IgE antibodies, such complexes probably also play a role in the induction of TNF in vivo. Overproduction of TNF is considered a major pathogenic mechanism responsible for fever and tissue lesions in P. falciparum malaria. This overproduction is generally assumed to reflect a direct stimulation of effector cells by certain parasite-derived toxins. Our results suggest that IgE elevation constitutes yet another important mechanism involved in excessive TNF induction in this disease.