Regulation of the immune response in Plasmodium falciparum malaria. II. Antigen specific proliferative responses in vitro.

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 et al., 1983). The malarial antigens used were sonicated membrane preparations or purified and concentrated supernatants from in vitro cultures of P. falciparum; 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. falciparum 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 cases, 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 response 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.     

Effects of autologous plasma on lymphocyte transformation in malaria and in acute protein-energy malnutrition. Comparison of purified lymphocyte and whole blood cultures.

Phytohaemagglutinin (PHA) induced lymphocyte transformation in whole blood and in purified lymphocyte cultures was investigated in Gambian children with acute Plasmodium falciparum malaria or with acute protein-energy malnutrition (PEM). Responses of purified lymphocytes cultured in the absence of autologous plasma were normal, with one exception. Autologous plasma depressed the response of purified lymphocytes to a low dose of PHA in several malaria and PEM patients. In whole blood cultures of 1 day and of 3 day duration, responses of several children with malaria or PEM were less than those of control children. Responses were not related to absolute lymphocyte counts. In 3 day, but not 1 day, cultures from control and malarious children, responses were inversely proportional to neutrophil counts. Cultures of whole blood and of purified lymphocytes in autologous plasma gave comparable results in 58 of 70 patients.     

Regulation of the immune response in Plasmodium falciparum malaria: IV. T cell dependent production of immunoglobulin and anti-P. falciparum antibodies in vitro.

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:1B) 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 (greater than 10 micrograms/ml). Neither of the antigens induced IgG secretion at concentrations of less than or equal to 10 micrograms/ml in control cultures of lymphocytes from patients with P. vivax 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 to induce T cell dependent responses in P. falciparum malaria.     

Parasite-derived mitogenic activity for human T cells in Plasmodium falciparum continuous cultures.

Supernatants from Plasmodium falciparum continuous cultures exhibited mitogenic activity against human blood lymphocytes from unsensitized donors. This effect, which was not observed with supernatants from control cultures grown in the absence of the parasites, was dependent upon (i) the concentration of supernatant added to the lymphocyte cultures and (ii) the parasite concentration in the P. falciparum continuous cultures. T cells were the predominant target cells of this mitogenic activity. We observed similar response in lymphocytes from malaria-sensitized individuals to P. falciparum continuous culture material. We also detected a mitogenic activity in parasite-infected erythrocytes from P. falciparum continuous cultures. P. falciparum continuous cultures may provide practical quantities of parasite-derived substances which, presumably, are able to manipulate the immune effector mechanisms of an infected host.     

Plasmodium falciparum induces apoptosis in human mononuclear cells.

The level of spontaneous apoptosis in short-term lymphocyte cultures was evaluated in different human immunodeficiency virus-negative groups of either healthy control individuals or patients with clinical malaria. The mean percentage of spontaneous apoptosis found in patients during a malaria attack was significantly higher than in sex- and age-matched healthy controls. The healthy asymptomatic controls were individuals with different degrees of exposure to Plasmodium falciparum as reflected by their various mean levels of specific anti-P. falciparum (immunoglobulin G and M) antibodies. The percentages of apoptotic nuclei were found to be significantly higher in lymphocytes from subjects living in an area where malaria is holoendemic than in lymphocytes from subjects less exposed. Concentrations of soluble plasma interleukin-2 receptor were also higher in subjects from areas where malaria is endemic than in other groups, revealing different levels of lymphocyte activation. Of particular relevance to the in vivo situation, a P. falciparum schizont-rich extract induced a systematic and significant elevation of apoptotic nuclei at day 6 in 87.5% (35 of 40) of the subjects tested. In additional studies with different concentrations of extract, [3H]thymidine incorporation was concomitant with a low or limited level of apoptosis. Taken together, our results strongly suggest that acute as well as chronic asymptomatic P. falciparum infections were consistently associated with a marked increase in the level of mononuclear cell apoptosis. This process could be implicated in some of the alterations reported for the proliferative T-cell responses in areas where malaria is endemic.     

Human antibodies against Plasmodium falciparum liver-stage antigen 3 cross-react with Plasmodium yoelii preerythrocytic-stage epitopes and inhibit sporozoite invasion in vitro and in vivo

The Plasmodium falciparum liver-stage antigen 3 (LSA3), a recently identified preerythrocytic antigen, induces protection against malaria in chimpanzees. Using antibodies from individuals with hyperimmunity to malaria affinity purified on recombinant or synthetic polypeptides of LSA3, we identified four non-cross-reactive B-cell epitopes in Plasmodium yoelii preerythrocytic stages. On sporozoites the P. yoelii protein detected has a molecular mass similar to that of LSA3. T-cell epitopes cross-reacting with P. yoelii were also demonstrated using peripheral blood lymphocytes from LSA3-immunized chimpanzees. In contrast, no cross-reactive epitopes were found in Plasmodium berghei. LSA3-specific human antibodies exerted up to 100% inhibition of in vitro invasion of P. yoelii sporozoites into mouse hepatocytes. This strong in vitro activity was reproduced in vivo by passive transfer of LSA3 antibodies. These results indicate that the homologous epitopes may be biologically functional and suggest that P. yoelii could be used as a model to assess the antisporozoite activity of anti-LSA3 antibodies.     

Naturally acquired human antibodies which recognize the first epidermal growth factor-like module in the Plasmodium falciparum merozoite surface protein 1 do not inhibit parasite growth in vitro.

Merozoite surface protein 1, one of the major surface proteins of the invasive blood stage of the malaria parasite, is a prime candidate for the development of a vaccine against the human disease. Previously, monoclonal antibodies which both inhibited the growth of Plasmodium falciparum in vitro and bound to the first of two epidermal growth factor-like modules located near the carboxy terminus of the protein had been identified. In this study, we have used affinity chromatography on a recombinant fusion protein corresponding to the first epidermal growth factor-like module in P. falciparum merozoite surface protein 1 to prepare antibody induced by natural infection. The antibody was purified from the total immunoglobulin G fraction of adult West African donors, shown to passively confer immunity against falciparum malaria. Such affinity-purified antibodies were shown to recognize the native protein by a number of separate criteria and to block the binding of an inhibitory monoclonal antibody, but they failed to inhibit parasite invasion in an in vitro growth assay. These results indicate that antibody alone is not sufficient to interfere with erythrocyte invasion.     

Plasmodium falciparum schizont sonic extracts suppress lymphoproliferative responses to mitogens and antigens in malaria-immune adults.

Cellular immune responses to malaria antigens are suppressed during acute Plasmodium falciparum infection, and evidence from both murine and human studies suggests that parasite-derived factors may be directly immunosuppressive. In this study we have shown that P. falciparum schizont sonic extract will suppress in vitro lymphoproliferative responses to purified malaria antigens and other soluble antigens. The degree of suppression appears to correlate with the level of the lymphoproliferative response to the schizont preparation and is correspondingly more marked in malaria-immune donors than in nonimmune individuals. The effect can be transferred with primed mononuclear cells and is partially abrogated by removal of CD8+ lymphocytes. The suppressive component of the schizont preparation is nondialyzable and partially heat labile and comigrates with hemoglobin-derived proteins in the molecular mass range 10 to 20 kilodaltons.     

Generation of Reactive Oxygen Radicals by Human Phagocytic Cells Activated by Plasmodium falciparum

The role of monocytes, macrophages and neutrophils in killing malaria parasites is well documented, and their involvement in malaria pathology has been suggested. However, the underlying mechanisms are not clear. The present study reports on the role of P. falciparum-parasitized erythrocytes, free merozoites, and culture supernatant antigens in the generation of reactive oxygen radicals by human peripheral blood monocytes and neutrophils. Blood neutrophils and monocytes obtained from healthy individuals were isolated by density gradient separation. A human isolate of P. falciparum was grown in continuous culture. Parasitized erythrocytes and free merozoites were prepared from synchronized cultures. Soluble antigens from culture supernatants were purified by affinity chromatography using CNBr-Sepharose 4B columns bound to specific IgG. Oxidative burst response of neutrophils and monocytes were determined by oxygen consumption, superoxide production, and chemiluminescence. It was found that P. falciparum merozoites and the soluble antigens were capable of activating neutrophils and monocytes in vitro and resulting in the production of oxygen radicals by these cells. In conclusion, these findings demonstrate that malaria antigens are able to activate normal human blood phagocytes and result in generation of oxygen radicals by these cells. The released oxygen radicals can then contribute to both the destruction of the parasite and the pathology of malaria.     

Epitopes recognized by human T lymphocytes on malaria circumsporozoite protein

The circumsporozoite protein of the malaria parasite Plasmodium falciparum contains regions of nonrepetitive sequences which are predicted to be T cell recognition sites. We synthesized peptides corresponding to three of these regions, and tested their ability to stimulate proliferation of peripheral blood lymphocytes from donors living in a malaria-endemic area, or from nonimmune donors. Cells from 15 out of 22 donors (including 4 of 6 nonimmune individuals) were stimulated by one or more of the peptides. T cell clones specific for one of the peptides were obtained and shown to recognize the native protein purified from sporozoites. These data help to identify T cell epitopes which could be incorporated into a malaria vaccine.     

Cell-mediated immune responses to Plasmodium falciparum antigens in adult Gambians.

Peripheral blood mononuclear cells from clinically immune Gambian adults were assayed for in vitro proliferation in response to crude and partially purified Plasmodium falciparum antigens. Lymphoproliferative responses to malaria antigens, lectin mitogens and Candida albicans were compared with those of control donors with no previous exposure to malaria. Cells of malaria-immune individuals were significantly more responsive to conconavalin A, and less responsive to phytohaemagglutinin, than cells from the control donors in both non-immune human serum and autologous serum. Cells from a proportion of immune donors proliferated in response to soluble malaria antigens but a substantial minority did not. Young adults and women were over-represented in the non-responding population. Responses to soluble malaria antigens were depressed in autologous serum compared with normal human serum. Both immune and control cells produced low levels of gamma-IFN when stimulated with crude P. falciparum schizont antigens. Approximately half the immune donors, and none of the controls, produced significant levels of gamma-IFN in response to purified soluble malaria antigen or malaria parasite culture supernatant. There was no direct correlation between lymphoproliferation and gamma-IFN production.     

肌球蛋白活化的T淋巴细胞诱导心肌自身免疫反应

目的研究心肌肌球蛋白活化的T淋巴细胞能否介导心肌的自身免疫反应。方法分离、提纯大鼠脾脏树突状细胞(DC)和T淋巴细胞,分为两组在体外混合培养活化组加入大鼠心肌肌球蛋白;未活化组不加大鼠心肌肌球蛋白。将活化组或未活化组的T淋巴细胞分别经尾静脉转输入大鼠体内。在转输后3d、1周和4周末处死大鼠,观察心肌的病理改变。结果转输活化T细胞的大鼠,于转输后3d即出现心肌的淋巴细胞浸润,1周末达到高峰伴有少许心肌坏死,4周末心肌的淋巴细胞浸润明显减轻,各组间差异有显著性(P<0.01)。转入活化T细胞大鼠的肝、肺、肾、脑和甲状腺等组织均未见淋巴细胞浸润。转输未活化T淋巴细胞的大鼠心肌未见淋巴细胞浸润。结论肌球蛋白活化的T淋巴细胞能够介导心肌的自身免疫反应。     

Malaria associated apoptosis is not significantly correlated with either parasitemia or the number of previous malaria attacks

The occurrence and intensity of lymphocyte apoptosis in blood samples from 79 outclinic patients with uncomplicated Plasmodium falciparum or Plasmodium vivax malaria and 30 healthy individuals were investigated. No difference in apoptosis percentages was detected between healthy individuals and malaria patients when ex vivo lymphocytes were analyzed. However, significantly increased apoptosis levels were observed in lymphocytes from both P. falciparum- and P. vivax-infected patients when the cells were cultured for 24 h. CD4(+)and CD8(+) T cells were affected to a comparable extent in P.falciparum- and P.vivax-infected patients. However, when we compared apoptosis values in infected and non-infected individuals it appeared that CD4(+) T cells were more susceptible than CD8(+) T cells. A significant increase in the sIL-2R plasma levels was observed in malaria patients when compared with healthy individuals and a positive correlation was observed between sIL-2R levels and apoptosis rates in infected patients presenting increased rates of apoptosis. An increased expression of Fas antigen was recorded after stimulation with P. falciparum antigen or anti-CD3 monoclonal antibody. These data show that a consistent proportion of the lymphocyte population dies by apoptosis during a malaria infection and that a period of time is necessary before in vivo activated cells can express the apoptotic process in vitro.     

Association between human serum-induced crisis forms in cultured Plasmodium falciparum and clinical immunity to malaria in Sudan.

Clinical histories with regard to falciparum malaria were collected from adults living in holo-, hyper-, and hypoendemic areas of Sudan and matched to serum samples which were assayed for antiparasitic activity in cultures of Plasmodium falciparum. The adult population of the endemic areas could be divided into three groups based on oral histories: those who never experience falciparum malaria; those with a childhood history of malaria, who experience only mild occasional malaria as adults; and those who suffer serious recurring malaria symptoms. In vitro parasite inhibition was greatest with sera from individuals with no clinical histories of malaria, and generally, more inhibition was noted in sera from holoendemic versus hyperendemic areas. Serum from hypoendemic urban Khartoum was not inhibitory. There was no relationship between serum indirect fluorescent antibody titers and parasite inhibition, but there was strong association between clinical immunity and intraerythrocytic parasite inhibition resulting in "crisis" forms. Purified immunoglobulin G was not strongly associated with crisis forms, which were consistently associated with fractions of immune serum remaining after immunoglobulin removal. Thus, it appears that clinical immunity to malaria in Sudan is based on nonantibody serum factors, possibly associated with cell-mediated immunity. Human leukocyte alpha-interferon had no inhibitory effects on cultured P. falciparum. Some umbilical cord sera were profoundly inhibitory, producing crisis forms, whereas others were not inhibitory, suggesting that factors that induce crisis forms may play a role in protecting neonates from falciparum malaria.     

Strain variation in tumor necrosis factor induction by parasites from children with acute falciparum malaria.

A small proportion of individuals infected with Plasmodium falciparum develop cerebral malaria. Why it affects some infected individuals but not others is poorly understood. Since tumor necrosis factor (TNF) has been implicated strongly in the pathogenesis of cerebral malaria, here we have compared different parasite isolates for their ability to induce TNF production by human mononuclear cells in vitro. Wild isolates were collected from 34 Gambian children with cerebral malaria and 66 children with uncomplicated malaria fever. Cerebral malaria isolates tended to stimulate more TNF production than mild malaria isolates, but there was considerable overlap between the two groups, and the present data provide only limited support for the hypothesis that cerebral malaria is caused by strains of P. falciparum inducing high levels of TNF. However, it is notable that the amounts of TNF induced by different wild isolates from a single locality differed by over 100-fold. The biological significance of this polymorphism deserves further scrutiny in view of the central role that TNF is believed to play in host defense and in the clinical symptomatology of human malaria.     

Tumor necrosis factor does not induce Plasmodium falciparum crisis forms.

Mouse and rabbit sera from animals treated with Mycobacterium bovis BCG and lipopolysaccharide contained tumor necrosis factor (TNF) and induced malaria parasite crisis forms. However, neither purified mouse- nor recombinant DNA-produced human TNF induced crisis forms in cultured Plasmodium falciparum. Furthermore, rabbit polyclonal and mouse monoclonal antibodies against human TNF did not block the parasite inhibitory activity of human malaria crisis form factor serum from Sudan.     

Inhibition of malaria parasite invasion of human erythrocytes by a lymphocyte membrane polypeptide.

Extraction by boiling of the buffy coat of human blood yields a protein solution which inhibits the propagation of the human malaria parasite Plasmodium falciparum in culture with a 50% inhibitory dose of 105 micrograms of protein per ml. The inhibitory activity is associated exclusively with the lymphocytes and affects solely the invasion of erythrocytes by free merozoites. Boiled extracts of isolated lymphocytes had a 50% inhibitory dose of 22 micrograms/ml. Fractionation of surface-labeled or pronase-treated lymphocytes revealed that the antimalarial lymphocyte factor is associated with the intracellular aspect of the membrane fraction and is probably not involved in the host defense system against malaria. Further purification by salt extraction, ion-exchange chromatography, molecular gel filtration, and electroelution from lithium dodecyl sulfate-polyacrylamide gels resulted in 300- to 550-fold purification, i.e., a 50% inhibitory dose of 40 to 70 ng/ml. All inhibitory fractions contained a 48-kilodalton polypeptide which eluted from a gel filtration column as a 400-kilodalton species, implying multimeric association. Some 6,000 molecules of the 48-kilodalton polypeptide bind with high affinity to one merozoite, the free form of the parasite. The Kd of 0.1 to 0.5 nM for the binding of the 48-kilodalton polypeptide correlated well with the 50% inhibitory dose of 0.3 to 0.4 nM obtained with purified active antimalarial lymphocyte factor. We therefore suggest that the 48-kilodalton polypeptide partially purified from lymphocyte membranes is the antimalarial lymphocyte factor and that it exerts its inhibitory activity by binding to merozoites, thereby preventing their invasion into erythrocytes. The antimalarial lymphocyte factor or a polypeptide sequence thereof could serve for further probing of invasion at the molecular level.     

The clinical-grade 42-kilodalton fragment of merozoite surface protein 1 of Plasmodium falciparum strain FVO expressed in Escherichia coli protects Aotus nancymai against challenge with homologous erythrocytic-stage parasites

A 42-kDa fragment from the C terminus of major merozoite surface protein 1 (MSP1) is among the leading malaria vaccine candidates that target infection by asexual erythrocytic-stage malaria parasites. The MSP1(42) gene fragment from the Vietnam-Oak Knoll (FVO) strain of Plasmodium falciparum was expressed as a soluble protein in Escherichia coli and purified according to good manufacturing practices. This clinical-grade recombinant protein retained some important elements of correct structure, as it was reactive with several functional, conformation-dependent monoclonal antibodies raised against P. falciparum malaria parasites, it induced antibodies (Abs) that were reactive to parasites in immunofluorescent Ab tests, and it induced strong growth and invasion inhibitory antisera in New Zealand White rabbits. The antigen quality was further evaluated by vaccinating Aotus nancymai monkeys and challenging them with homologous P. falciparum FVO erythrocytic-stage malaria parasites. The trial included two control groups, one vaccinated with the sexual-stage-specific antigen of Plasmodium vivax, Pvs25, as a negative control, and the other vaccinated with baculovirus-expressed MSP1(42) (FVO) as a positive control. Enzyme-linked immunosorbent assay (ELISA) Ab titers induced by E. coli MSP1(42) were significantly higher than those induced by the baculovirus-expressed antigen. None of the six monkeys that were vaccinated with the E. coli MSP1(42) antigen required treatment for uncontrolled parasitemia, but two required treatment for anemia. Protective immunity in these monkeys correlated with the ELISA Ab titer against the p19 fragment and the epidermal growth factor (EGF)-like domain 2 fragment of MSP1(42), but not the MSP1(42) protein itself or the EGF-like domain 1 fragment. Soluble MSP1(42) (FVO) expressed in E. coli offers excellent promise as a component of a vaccine against erythrocytic-stage falciparum malaria.     

Opsonic activity of human immune serum on in vitro phagocytosis of Plasmodium falciparum infected red blood cells by monocytes.

In vitro human monocytes from normal blood donors ingest red blood cells infected with Plasmodium falciparum more efficiently than normal red blood cells (NRBC). The phagocytic activity of human monocytes for infected red blood cells (IRBC) is greatly enhanced by the addition of immune sera obtained from individuals living in areas with endemic malaria. In contrast, the addition of sera obtained from individuals recovering from a first infection, or pooled normal sera, does not result in increased phagocytosis of IRBC. The phagocytosis enhancing activity of immune sera is associated with the IgG fraction and IgG depleted sera do not stimulate phagocytosis. Enhanced immune serum mediated phagocytosis occurs as a result of opsonization of IRBC. This was demonstrated by experiments in which monocytes or IRBC were preincubated with immune serum prior to the phagocytic assay. The opsonic activity could be absorbed by IRBC but not by NRBC. The opsonization of IRBC and subsequent phagocytosis were also dependent on the stage of development of the intracellular parasite. IRBC containing schizonts and trophozoites were preferentially phagocytosed as compared with ring forms. The role of malaria induced surface alterations and/or malaria surface antigens in the opsonization of IRBC by immune sera is discussed. These experiments suggest that phagocytosis of P. falciparum IRBC by monocytes may play a role in the immune elimination of malaria infection in humans.     

Isolation of a Plasmodium falciparum rhoptry protein

A monoclonal antibody raised against the malaria parasite Plasmodium falciparum recognised a protein of 140000 molecular weight which was synthesized during schizogony. The protein has been purified by monoclonal antibody affinity chromatography from extracts of parasitized red cells. Antibodies against the protein have been used to determine its subcellular location. The protein is not expressed on the merozoite surface and has been located in the rhoptries, the apical organelles of the merozoite.