Characterization of the spleen B-cell compartment at the early and late blood-stage Plasmodium chabaudi malaria.

Polyclonal B-cell activation is a feature of the early spleen cell response to blood-stage Plasmodium chabaudi malaria. Immunity to blood-stage malaria is guaranteed by the generation of B cells able to produce parasite-specific antibodies mainly from the immunoglobulin (Ig)G2a isotype. In the present study, we characterized the spleen B-cell compartment during blood-stage P. chabaudi infection. The numbers of B220(+) and B220(LOW) CD138(+) (plasma) cells increased sharply between days 4 and 7 post-infection (p.i.). At this time B220(+) cells expressed surface (s)IgM, but nearly all B220(LOW) CD138(+) cells showed concomitantly intracellular (i)IgM and IgG2a. Both follicular and marginal zone B cells were activated expressing high amounts of CD69. At day 40 p.i., B220(LOW) CD138(+) cell population was still increased but, differently from acute infection, 61.1% of these cells were positive for iIgG2a while only 14.2% expressed iIgM. Moreover, at days 20 and 40 p.i., 29.2% and 13.0% of B220(+) cells expressed sIgG2a, respectively. According to cell size and expression of CD80, CD86, CD11b, CD44 and CD38, B220(+) sIgG2a(+) cells had a phenotype characteristic of activated/memory B cells. Furthermore, 14.1% of B220(+) sIgG2a(+) cells at day 30 p.i. expressed a marginal zone B-cell phenotype. Importantly, B cells from 40-day-infected mice were very efficient in presenting parasite antigens leading to proliferation of both CD4(+) and CD8(+) cells. Our results contribute for understanding the dynamics of B cells during P. chabaudi infection, underlying the mechanisms of antigen presentation and antibody production, which are essential for the acquisition of protective immunity against malaria.

Role of CD28 in polyclonal and specific T and B cell responses required for protection against blood stage malaria.

The role of B7/CD28 costimulatory pathway in the polyclonal and specific lymphocyte activation induced by blood stages of Plasmodium chabaudi AS was investigated in CD28 gene knockout (CD28(-/-)) and C57BL/6 (CD28(+/+)) mice. Analysis of the spleen during the acute infection revealed a similar increase in T and B cell populations in both groups of mice. Moreover, CD28(-/-) mice were able to develop a polyclonal IgM response to P. chabaudi. On the contrary, the polyclonal IgG2a response was markedly reduced in the absence of CD28. Production of IFN-gamma; up-regulation of CD69, CD40L, CD95 (Fas), and CD95L (Fas ligand); and induction of apoptosis were also affected by the lack of CD28. Interestingly, the ability to control the first parasitemia peak was not compromised in acutely infected CD28(-/-) mice, but CD28(-/-) mice failed to eradicate the parasites that persisted in the blood for >3 mo after infection. In addition, drug-cured CD28(-/-) mice were unable to generate memory T cells, develop an anamnesic IgG response, or eliminate the parasites from a secondary challenge. The incapacity of CD28(-/-) mice to acquire a full protective immunity to P. chabaudi correlated with an impaired production of specific IgG2a. Moreover, reinfected CD28(-/-) mice were protected by the adoptive transfer of serum from reinfected CD28(+/+) mice containing specific IgG2a. Our results demonstrate that the polyclonal lymphocyte response is only partially affected by the absence of CD28, but this coreceptor is essential to generate specific T and B cell responses required for complete protection against P. chabaudi malaria.

Susceptibility of the different developmental stages of the asexual (schizogonic) erythrocyte cycle of Plasmodium chabaudi chabaudi to hyperimmune serum, immunoglobulin (Ig)G1, IgG2a and F(ab')2 fragments.

The mechanisms by which antibodies interfere with Plasmodium growth are still under debate. Characterizing the asexual erythrocyte stages susceptible to antibodies from hyperimmune individuals is therefore a relevant contribution to vaccine research. In this study, using a virulent and synchronous murine malaria parasite, Plasmodium chabaudi chabaudi AJ, we have shown that trophozoites and circulating schizonts are not the main targets for antibodies from hyperimmune serum. In drug-cured mice challenged with a high inoculum of ring-infected erythrocytes, parasitemias do not decline until the moment of erythrocyte rupture, suggesting that effector mechanisms operate immediately prior to reinvasion. Confirming these findings, treatment of primary-infected mice with hyperimmune serum inhibited the generation of new ring forms, but did not alter the numbers of schizont-infected erythrocytes, despite the fact that these cells were recognized by immunoglobulin (Ig)G antibodies. When these mice were treated with IgG1 or IgG2a purified from hyperimmune serum, both subclasses limited reinvasion, but IgG2a showed a stronger protective activity. The fact that Fc digestion decreases but does not abrogate protection suggests that both Fc-dependent and independent mechanisms participate in this process. Treatment with cobra venom factor did not interfere with the antibody-mediated protection, ruling out the participation of the complement system in both lysis and phagocytosis of merozoites or infected erythrocytes. Therefore, in mice suffering from P. c. chabaudi AJ malaria, merozoite neutralization seems to be a major mechanism of protection conferred by hyperimmune serum antibodies. However, FcgammaR-mediated interactions, or other mechanisms not yet defined, may also contribute to inhibit erythrocyte reinvasion.

Calcium homeostasis in intraerythrocytic malaria parasites.

The fluorescent indicator, fura-2, AM, was used to measure free calcium concentrations in the intraerythrocytic malaria parasites of Plasmodium chabaudi and Plasmodium falciparum. In both species the free cytosolic calcium concentration was maintained at low levels (between 40 and 100 nM throughout the maturation process. Digital image analysis of the indicator fluorescence was performed on parasites and evaluated with the aid of a calibration of the calcium response, based on permeabilized parasites, exposed to calcium buffers. This again revealed that free calcium concentrations in the intact parasite are maintained at a predetermined level, regardless of the free calcium in the surrounding milieu. Both species of parasites are thus capable of regulating their internal free calcium levels with high precision, presumably by means of calcium pump ATPases. A small but significant elevation of the cytosolic free calcium concentration by the tumor promoter, thapsigargin, may be taken to reflect the presence of calcium stores in the endoplasmic reticulum in P. falciparum.

A megadalton protein (Pc2500) is present during the intraerythrocytic development of P. chabaudi.

Red cells infected with Plasmodium chabaudi display a megadalton protein, named Pc2500, synthesized during the early trophozoite stage with an apparent molecular mass of 2500 kDa. When infected red cells at the young trophozoite stage metabolically labeled with [35S] methionine are treated with Triton X-100, the megadalton protein remains in the insoluble fraction. At this stage, Pc2500 was found to be phosphorylated when infected red cells were incubated with [32P] orthophosphate. In late trophozoite stage, the Pc2500 is solubilized in Triton X-100 and is unphosphorylated. Infected cells, were subjected to nitrogen cavitation. Analysis of isolated erythrocyte membranes and parasites using electron microscopy and marker enzymes demonstrated the purity of these fractions. In the phosphorylated from, Pc2500 was shown to be mostly associated with the parasite whereas, at the late trophozoite stage, it was found to have migrated to the host cell membrane.