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It has earlier been reported that the human monoclonal antibody (MoAb 33G2) and polyclonal antibodies reactive with Pf332 may interfere in vitro with the erythrocytic cycle of Plasmodium falciparum at two potential target sites for protective antibodies, indicating that the antigen may constitute an important target for immune responses during malaria infections. MoAb 33G2 shows its highest reactivity with repeated sequences in the antigen Pr332 and also cross-reacts with determinants in Pf155/RESA. This study was conducted in order to assess the prevalence of seroreactivity against Pf332 in individuals residing in areas of different malaria endemicity, and in children with different degrees of disease severity. We now report that individuals resident in malaria endemic regions show a high prevalence of seroreactivity to antigen Pf332 repeat sequences. The mean antibody concentrations were significantly higher in donors from Liberia. Madagascar and Gambia compared with Thai and Colombian donors, probably reflecting the higher degree of exposure in the African regions. Although the levels of such antibodies in individual sera correlated well with the levels of antibodies to one Pf155/RESA repeat peptide, only a minor part of the peptidereactive antibodies were cross-reactive between the two antigens. In Gambian children, the mean concentrations of antibodies reactive with PD32 or Pf 155/RESA peptides were significantly higher in children with severe than with mild malaria. Further longitudinal studies are needed to evaluate the capacity of Pf332 to induce potentially protective or harmful antibody responses. [ABSTRACT FROM AUTHOR]

Repeat sequences from the Plasmodium falciparum blood stage antigen Pf332 frequently comprise the pentapeptide VTEEI, an epitope recognized by certain parasite neutralizing antibodies. This B-cell epitope was assembled in an octavalent multiple antigen peptide (MAP) system either as trimers (VTEEI)3 (MAP1) or as an integral part of a naturally occurring Pf332 undecamer repeat sequence SVTEEIAEEDK (MAP2). Characteristics of the immunogenicity of these subunit constructs were evaluated in H-2 congenic mice. MAP1 generated antibody responses in mice of the H-2d, H-2k and H-2q haplotypes, but not in H-2b or H-2s mice, whereas MAP2 only induced antibodies in mice of H-2k haplotype. When analysing T-cell responses induced by the MAP, lymph node cells from responder strains primed in vivo with MAPI proliferated in response to restimulation with both MAPI and the peptide (VTEEI)3. MAP2, however, did not induce a detectable T-cell proliferation. Additionally, the lack of antibody response to MAP1 in H-2b mice could be circumvented by combining the MAP1 peptide and a H-2b-restricted T-cell epitope in a diepitope MAP construct. Despite the fact that the motif VTEEI has not been identified in Pf332 sequences in the form of a trimer, MAP1 did induce Pf332 protein-reactive antibodies. Assembly of multimers of short defined epitopes in MAP constitutes an interesting approach for the design of polyvalent subunit immunogens. [ABSTRACT FROM AUTHOR]

Antigenic diversity in held 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 front 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. [ABSTRACT FROM AUTHOR]

An enzyme linked immunosorbent assay (ELISA) has been developed to estimate disease related antibodies in sera from malaria patients or individuals living in malaria endemic areas. As antigen, Percoll enriched fractions (mainly late trophozoites, schizonts) from Plasmodium falciparum in vitro cultures were used. An ELISA with ghosts from normal human red blood cells (RBC) was performed in parallel. One hundred and seventy-five sera were tested for their reactivity with either one of the two antigens. Seven sera from patients with acute P.falciparum infection were negative. Most of these had been taken very early in infection and consecutive samples taken later usually were positive. The antibodies reacting with the P. falciparum antigen had a high parasite specificity as indicated by inhibition and absorption experiments. Many sera also had elevated levels of antibodies specific for RBC antigens. A correlation, most pronounced in the IgM system, was also seen between the anti-RBC and the -duti-P. falciparum antibody levels. [ABSTRACT FROM AUTHOR]

The IgG subclass levels of anti-Plasmodium falciparum antibodies in human sera were determined in ELISA with monoclonal mouse antibodies specific for the human IgG subclasses as analytical reagents. The parasite antigen was a trophozoite/schizont enriched preparation of in vitro cultivated P. falciparum. Serum samples were from Swedish malaria patients and adult Liberians. Parasite specific antibodies were found in all four subclasses. Relatively elevated levels of IgGl antibodies were found both in Swedish patients and in the Liberians. Relative to the Liberians, high IgG2 antibody levels were seen in most Swedish patients. In Liberian sera but not in those from Swedish patients elevated IgG3 levels were found. In two Swedish patients followed consecutively for a period of 15 weeks, elevated levels of IgG1 and IgG3 antibodies were seen after relapse. No differences between the groups in regard to the levels origG4 antibodies were noticed. The differences between the Swedish patients and the Liberians were also in part reflected by differences in the total amounts of the four IgG subclasses. [ABSTRACT FROM AUTHOR]

People living in Plasmodium falciparum-endemic areas frequently have elevated levels of total as well as P. falciparum-specific serum IgE. This study aimed at investigating whether the elevated serum IgE levels reflect a shift in the balance between CD4+ T helper 1 (Th1) and T helper 2 (Th2) cells in individuals naturally exposed to the P. falciparum parasite. To investigate the role of Thi and Th2 ceils in the human P. falciparum system we used the ELISPOT assay to determine the ratio of IEN-γ- and IL-4-producing cells after specific antigen or mitogen activation in vitro. The donors were individuals who had acquired immunity through natural exposure to the parasite. In response to the specific malaria antigens, very few IL-γ-producing cells were seen. However, in the response of individual donors to the polyclonal T cell activator, leucoagglutinin (La), the anti-malarial IgE levels in plasma were correlated with an increased ratio of IL-4/IFN-γ producing cells. Thus, donors with ratios of IL-4/IFN-γ>1 exhibited mean plasma anti-malarial IgE levels significantly greater than those with ratios <1. In individuals not living in P. fatciparum-endemic areas the ratio of IL-4/IFN-γ was always <1. Taken together, our data suggest a shift in the balance between Th1 and Th2 cells in naturally P. falciparum primed individuals, associated with elevated anti-P. fatciparum plasma IgE levels. The role and biological signilicance of IgE (Th2-type immune response) for protection against F. faiciparum and/or pathogenesis of malaria require further study. [ABSTRACT FROM AUTHOR]

T cells from patients with acute Plasmodium falciparum malaria were investigated for induction of immunoglobulin- or anti-malaria antibody secretion in vitro. Stimulation of autologous T/B cell mixtures (2T: IB) with low concentrations of P. falciparum antigen and cultured for 12 days gave rise to a T-dependent IgG secretion which was significantly elevated over that in medium controls. This was achieved with both a crude P. falciparum antigen and a partially purified preparation enriched in Pf 155. a merozoite-derived antigen deposited in the red cell membrane at invasion (Perlmann et al. 1984), Control antigen (RBC ghosts) induced IgG secretion only when added at high concentrations (>10 μg/ml). Neither of the antigens induced IgG secretion at concentrations of ≪ 10μg/ ml in control cultures of lymphocytes from patients with P. vita.\ malaria. Supernatants from cultures of P.falciparum patients frequently contained anti-P. falciparum antibodies when nanogram quantities (10-100 ng/ml) of either one of the two malaria antigen preparations was used for stimulation. No anti-P. falciparum antibodies were induced by the control antigen at any concentration. The induced anti-P. falciparum antibodies were directed to intracellular parasites and, at lower frequencies. to Pf 155 as detected on the surface of infected erythrocytes. The induction in vitro of anti-P. falciparum antibodies appeared to be correlated with the presence of such antibodies in the sera of the lymphocyte donors. The lymphocytes of only one out of eight P. vivax patients responded to antigen stimulation by secreting anti-P. falciparum antibodies. However, this donor (but not any of the others), was also P. falciparum seropositive. Taken together, these results indicate that the induction of anti-P. falciparum antibody secretion reflects a secondary response in vitro of cells primed in vivo. The present experimental system should be well suited to map parasite antigen for their capacity lo induce T cell dependent responses in P.falciparum malaria. [ABSTRACT FROM AUTHOR]

The antigen-induced DNA synthesis in vitro in lymphocytes from patients with acute Plasmodium falciparum malaria was investigated. The patients and healthy controls from Sweden or Colombia were the same as those studied in the accompanying paper (Troye-Blomberg el al., 1983). The malarial antigens used were sonicated membrane preparations or purified and concentrated supernatants from in vitro cultures of P. facliparum, similar preparations derived from normal human erythrocytes served as control antigen. In the patients' lymphocytes P. falciparum antigens induced a weak or moderate but significant stimulation of DNA synthesis, peaking after 3-4 days of incubation. This early response was specific for P. faleiparum since it was not obtained with lymphocytes from healthy donors nor with those from patients with acute P. vivax or P. ovale malaria. No antigen-induced response was seen in about half of the P.falciparum patients. However in a few negative eases, available for consecutive testing, positive reactions were seen with lymphocytes taken 2 weeks after infection when the blood of these patients was free of parasites. The early response induced in patients' lymphocytes to the P. falciparum antigens was not obtained with RBC antigen, however, these preparations frequently induced a response rising to significant levels later during incubation (day 5-6)- Similar delayed responses were obtained when either- patients' or control donors' lymphocytes were exposed to the P. falciparum antigens. This indicates that both the RBC and the parasite preparations contained mitogenic substances affecting human lymphocytes in general and easily obscuring the P, falciparum specific responses seen only in the patients. This latter response was relatively low and short lived, suggesting that it reflected a secondary in vitro stimulation of in vivo primed lymphocytes and that it was regulated by suppressor mechanisms. [ABSTRACT FROM AUTHOR]

The mitogen-induced DNA synthesis in vitro in lymphocytes from 20 patients acutely ill with Plasmodium falciparum malaria was compared with that of 16 healthy donors. Within both groups part of the donors were individuals who had only experienced short exposure or none at all to the parasite (Sweden) while the other part were donors living in a malaria endemic area (Colombia), The proliferative response to the T cell mitogen La (leucoagglutinin from PHA) of the patients was significantly reduced as compared with that of the controls. With pokeweed mitogen which stimulates T cells and induces a T cell-dependent activation of B cells, no difference between patients or controls was seen. The results were similar for the donors of different geographical origin and malaria background. Lymphocytes and monocytes from the peripheral blood of these donors were also studied for surface marker distribution by means of monoclonal antibodies. Both the absolute and the relative frequencies of T cells in the blood of the malaria patients were significantly reduced as compared with the controls. Furthermore, in almost all eight patients tested, the ratio between T4+ T cells (including the helper/inducer subsets) and T8+ T cells (including the suppressor and cytotoxic subsets) were below 1: 1 while they were close to 2:1 in the controls. The results indicate that the relative frequency of T8+ T cells, expressed as percentage total T cells (T.3+ ) was significantly elevated in the P. falciparum patients. The possible relationship between this imbalance and the irregular La response of the patients lymphocytes requires further investigation of lymphocyte function. [ABSTRACT FROM AUTHOR]