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Antibodies that inhibit malaria merozoite surface protein-1 processing and erythrocyte invasion are blocked by naturally acquired human antibodies.

Guevara Patiño JA, Holder AA, McBride JS, Blackman MJ - J. Exp. Med. (1997)

Bottom Line: Most significantly, affinity-purified, naturally acquired human antibodies specific for epitopes within the NH2-terminal 83-kD domain of MSP-1 very effectively block the processing-inhibitory activity of the anti-MSP-119 mAb 12.8.Blocking antibodies therefore (a) are part of the human response to malarial infection; (b) can be induced by MSP-1 structures unrelated to the MSP-119 target of processing-inhibitory antibodies; and (c) have the potential to abolish protection mediated by anti-MSP-119 antibodies.Our results suggest that an effective MSP-119-based falciparum malaria vaccine should aim to induce an antibody response that prevents MSP-1 processing on the merozoite surface.

View Article: PubMed Central - PubMed

Affiliation: Division of Parasitology, National Institute for Medical Research, London, United Kingdom.

ABSTRACT
Merozoite surface protein-1 (MSP-1) of the human malaria parasite Plasmodium falciparum undergoes at least two endoproteolytic cleavage events during merozoite maturation and release, and erythrocyte invasion. We have previously demonstrated that mAbs which inhibit erythrocyte invasion and are specific for epitopes within a membrane-proximal, COOH-terminal domain of MSP-1 (MSP-119) prevent the critical secondary processing step which occurs on the surface of the extracellular merozoite at around the time of erythrocyte invasion. Certain other anti-MSP-119 mAbs, which themselves inhibit neither erythrocyte invasion nor MSP-1 secondary processing, block the processing-inhibitory activity of the first group of antibodies and are termed blocking antibodies. We have now directly quantitated antibody-mediated inhibition of MSP-1 secondary processing and invasion, and the effects on this of blocking antibodies. We show that blocking antibodies function by competing with the binding of processing-inhibitory antibodies to their epitopes on the merozoite. Polyclonal rabbit antibodies specific for certain MSP-1 sequences outside of MSP-119 also act as blocking antibodies. Most significantly, affinity-purified, naturally acquired human antibodies specific for epitopes within the NH2-terminal 83-kD domain of MSP-1 very effectively block the processing-inhibitory activity of the anti-MSP-119 mAb 12.8. The presence of these blocking antibodies also completely abrogates the inhibitory effect of mAb 12.8 on erythrocyte invasion by the parasite in vitro. Blocking antibodies therefore (a) are part of the human response to malarial infection; (b) can be induced by MSP-1 structures unrelated to the MSP-119 target of processing-inhibitory antibodies; and (c) have the potential to abolish protection mediated by anti-MSP-119 antibodies. Our results suggest that an effective MSP-119-based falciparum malaria vaccine should aim to induce an antibody response that prevents MSP-1 processing on the merozoite surface.

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Processing-inhibitory anti–MSP119  mAbs can prevent MSP-1 and erythrocyte invasion  in in vitro culture, and can be rendered ineffective  by the simultaneous presence of anti-pME6 blocking antibodies. (A) Dose–response effect of mAb  12.10 on MSP-1 secondary processing. Metabolically radiolabeled T9/96 schizonts were supplemented with fresh erythrocytes and medium to obtain a parasitemia of ∼2% and a hematocrit of 1%.  The culture was then divided into equal aliquots  and incubated at 37°C in the presence of 5 mM  EGTA as control inhibitor (lane 1), or mAb 12.10  at a final concentration of 2 μg ml−1 (lane 2), 1 μg  ml−1 (lane 3), 500 μg ml−1 (lane 4), 400 μg ml−1  (lane 5), 300 μg ml−1 (lane 6), 200 μg ml−1 (lane  7), 100 μg ml−1 (lane 8), or no antibody (lane 9).  Schizont rupture and merozoite release were then  allowed to proceed for 6 h, and culture supernatants were analyzed by immunoprecipitation using  mAb X509 coupled to Sepharose for the presence  of MSP-133. (B) Blocking anti-pME6 antibodies reverse the processing-inhibitory (top) and invasion-inhibitory (bottom) activity of mAb 12.8. Cultures  containing metabolically radiolabeled T9/96 schizonts prepared as described above were incubated in  the presence of 5 mM EGTA (lane 1), 10% (vol/ vol) nonimmune human serum (lane 2), anti-pME6  antibodies (lane 3), mAb 12.8 (lane 4), mAb 12.8  plus anti-pME6 antibodies (lane 5), mAb 12.10  (lane 6), mAb 12.10 plus anti-pME6 antibodies  (lane 7), mAb 89.1 (lane 8), mAb 89.1 plus mAb  12.8 (lane 9) and mAb 89.1 plus mAb 12.10 (lane 10). In this case all antibodies were added to a final concentration of 400 μg ml−1. Analysis of 6-h culture supernatants by immunoprecipitation with mAb X509 (B, top) was as above, and in addition erythrocyte invasion in individual cultures was assessed  by counting the number of ring-stage parasites in 5,000 red cells, in triplicate (B, bottom). Invasion is expressed as a percentage of the ring-stage parasitemia (10%) obtained in a control culture with no additions (data not shown).
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Figure 7: Processing-inhibitory anti–MSP119 mAbs can prevent MSP-1 and erythrocyte invasion in in vitro culture, and can be rendered ineffective by the simultaneous presence of anti-pME6 blocking antibodies. (A) Dose–response effect of mAb 12.10 on MSP-1 secondary processing. Metabolically radiolabeled T9/96 schizonts were supplemented with fresh erythrocytes and medium to obtain a parasitemia of ∼2% and a hematocrit of 1%. The culture was then divided into equal aliquots and incubated at 37°C in the presence of 5 mM EGTA as control inhibitor (lane 1), or mAb 12.10 at a final concentration of 2 μg ml−1 (lane 2), 1 μg ml−1 (lane 3), 500 μg ml−1 (lane 4), 400 μg ml−1 (lane 5), 300 μg ml−1 (lane 6), 200 μg ml−1 (lane 7), 100 μg ml−1 (lane 8), or no antibody (lane 9). Schizont rupture and merozoite release were then allowed to proceed for 6 h, and culture supernatants were analyzed by immunoprecipitation using mAb X509 coupled to Sepharose for the presence of MSP-133. (B) Blocking anti-pME6 antibodies reverse the processing-inhibitory (top) and invasion-inhibitory (bottom) activity of mAb 12.8. Cultures containing metabolically radiolabeled T9/96 schizonts prepared as described above were incubated in the presence of 5 mM EGTA (lane 1), 10% (vol/ vol) nonimmune human serum (lane 2), anti-pME6 antibodies (lane 3), mAb 12.8 (lane 4), mAb 12.8 plus anti-pME6 antibodies (lane 5), mAb 12.10 (lane 6), mAb 12.10 plus anti-pME6 antibodies (lane 7), mAb 89.1 (lane 8), mAb 89.1 plus mAb 12.8 (lane 9) and mAb 89.1 plus mAb 12.10 (lane 10). In this case all antibodies were added to a final concentration of 400 μg ml−1. Analysis of 6-h culture supernatants by immunoprecipitation with mAb X509 (B, top) was as above, and in addition erythrocyte invasion in individual cultures was assessed by counting the number of ring-stage parasites in 5,000 red cells, in triplicate (B, bottom). Invasion is expressed as a percentage of the ring-stage parasitemia (10%) obtained in a control culture with no additions (data not shown).

Mentions: These data clearly show that the binding of antibodies specific to one component of the MSP-1–derived merozoite surface complex can interfere with the binding of antibodies to another component of the complex. Erythrocyte invasion by the malaria merozoite is rapid, going to completion within seconds of the initial interaction between parasite and red cell surface (39). Over such a short time span, could the presence of blocking antibodies interfere with the ability of processing-inhibitory antibodies to bind the merozoite surface and prevent both processing and invasion? To address this question directly in an in vitro system, a series of invasion experiments was performed. Mature, biosynthetically radiolabeled T9/96 schizonts were washed and placed in culture with fresh red cells. Merozoite release and red cell invasion were then allowed to proceed in the presence or absence of mAbs 12.8 and 12.10, with or without the additional presence of affinity-purified anti-pME6 human antibodies. The overall efficiency of invasion was assessed by counting the number of new ring stage parasites formed over the course of the experiment; MSP-1 processing in individual samples was subsequently assessed by direct immunoprecipitation of MSP-133 from the culture supernatants using mAb X509 coupled to Sepharose. In preliminary dose–response experiments, a concentration of ⩾400 μg ml−1 of either mAb 12.10 (Fig. 7 A) or mAb 12.8 (data not shown) was sufficient to reduce the amount of MSP-133 release to a level of inhibition seen in the presence of 5 mM EGTA, a potent inhibitor of MSP-1 secondary processing (11). The results of a typical experiment (of a total of three independent experiments) investigating the effects of the anti-pME6 blocking antibodies on the activity of mAbs 12.8 and 12.10 are presented in Fig. 7 B. In isolation, mAbs 12.8 and 12.10 virtually abolished both invasion (Fig. 7 B, bottom) and MSP-133 release (Fig. 7 B, top). However, in the presence of equal concentrations of the anti-pME6 human antibodies, the effects of mAb 12.8, but not of 12.10, were completely reversed (Fig. 7 B, lanes 5 and 7). Neither the anti-pME6 antibodies alone nor mAb 89.1 alone had any effect on either processing or invasion (Fig. 7 B, lanes 3 and 8), and mAb 89.1 exhibited no blocking activity (Fig. 7 B, lanes 9 and 10). These results unambiguously demonstrate that, under conditions of active release of viable merozoites, mAbs 12.8 and 12.10 effectively prevent both MSP-1 processing and erythrocyte invasion, and this activity can be efficiently abrogated by the presence of human blocking antibodies.


Antibodies that inhibit malaria merozoite surface protein-1 processing and erythrocyte invasion are blocked by naturally acquired human antibodies.

Guevara Patiño JA, Holder AA, McBride JS, Blackman MJ - J. Exp. Med. (1997)

Processing-inhibitory anti–MSP119  mAbs can prevent MSP-1 and erythrocyte invasion  in in vitro culture, and can be rendered ineffective  by the simultaneous presence of anti-pME6 blocking antibodies. (A) Dose–response effect of mAb  12.10 on MSP-1 secondary processing. Metabolically radiolabeled T9/96 schizonts were supplemented with fresh erythrocytes and medium to obtain a parasitemia of ∼2% and a hematocrit of 1%.  The culture was then divided into equal aliquots  and incubated at 37°C in the presence of 5 mM  EGTA as control inhibitor (lane 1), or mAb 12.10  at a final concentration of 2 μg ml−1 (lane 2), 1 μg  ml−1 (lane 3), 500 μg ml−1 (lane 4), 400 μg ml−1  (lane 5), 300 μg ml−1 (lane 6), 200 μg ml−1 (lane  7), 100 μg ml−1 (lane 8), or no antibody (lane 9).  Schizont rupture and merozoite release were then  allowed to proceed for 6 h, and culture supernatants were analyzed by immunoprecipitation using  mAb X509 coupled to Sepharose for the presence  of MSP-133. (B) Blocking anti-pME6 antibodies reverse the processing-inhibitory (top) and invasion-inhibitory (bottom) activity of mAb 12.8. Cultures  containing metabolically radiolabeled T9/96 schizonts prepared as described above were incubated in  the presence of 5 mM EGTA (lane 1), 10% (vol/ vol) nonimmune human serum (lane 2), anti-pME6  antibodies (lane 3), mAb 12.8 (lane 4), mAb 12.8  plus anti-pME6 antibodies (lane 5), mAb 12.10  (lane 6), mAb 12.10 plus anti-pME6 antibodies  (lane 7), mAb 89.1 (lane 8), mAb 89.1 plus mAb  12.8 (lane 9) and mAb 89.1 plus mAb 12.10 (lane 10). In this case all antibodies were added to a final concentration of 400 μg ml−1. Analysis of 6-h culture supernatants by immunoprecipitation with mAb X509 (B, top) was as above, and in addition erythrocyte invasion in individual cultures was assessed  by counting the number of ring-stage parasites in 5,000 red cells, in triplicate (B, bottom). Invasion is expressed as a percentage of the ring-stage parasitemia (10%) obtained in a control culture with no additions (data not shown).
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Figure 7: Processing-inhibitory anti–MSP119 mAbs can prevent MSP-1 and erythrocyte invasion in in vitro culture, and can be rendered ineffective by the simultaneous presence of anti-pME6 blocking antibodies. (A) Dose–response effect of mAb 12.10 on MSP-1 secondary processing. Metabolically radiolabeled T9/96 schizonts were supplemented with fresh erythrocytes and medium to obtain a parasitemia of ∼2% and a hematocrit of 1%. The culture was then divided into equal aliquots and incubated at 37°C in the presence of 5 mM EGTA as control inhibitor (lane 1), or mAb 12.10 at a final concentration of 2 μg ml−1 (lane 2), 1 μg ml−1 (lane 3), 500 μg ml−1 (lane 4), 400 μg ml−1 (lane 5), 300 μg ml−1 (lane 6), 200 μg ml−1 (lane 7), 100 μg ml−1 (lane 8), or no antibody (lane 9). Schizont rupture and merozoite release were then allowed to proceed for 6 h, and culture supernatants were analyzed by immunoprecipitation using mAb X509 coupled to Sepharose for the presence of MSP-133. (B) Blocking anti-pME6 antibodies reverse the processing-inhibitory (top) and invasion-inhibitory (bottom) activity of mAb 12.8. Cultures containing metabolically radiolabeled T9/96 schizonts prepared as described above were incubated in the presence of 5 mM EGTA (lane 1), 10% (vol/ vol) nonimmune human serum (lane 2), anti-pME6 antibodies (lane 3), mAb 12.8 (lane 4), mAb 12.8 plus anti-pME6 antibodies (lane 5), mAb 12.10 (lane 6), mAb 12.10 plus anti-pME6 antibodies (lane 7), mAb 89.1 (lane 8), mAb 89.1 plus mAb 12.8 (lane 9) and mAb 89.1 plus mAb 12.10 (lane 10). In this case all antibodies were added to a final concentration of 400 μg ml−1. Analysis of 6-h culture supernatants by immunoprecipitation with mAb X509 (B, top) was as above, and in addition erythrocyte invasion in individual cultures was assessed by counting the number of ring-stage parasites in 5,000 red cells, in triplicate (B, bottom). Invasion is expressed as a percentage of the ring-stage parasitemia (10%) obtained in a control culture with no additions (data not shown).
Mentions: These data clearly show that the binding of antibodies specific to one component of the MSP-1–derived merozoite surface complex can interfere with the binding of antibodies to another component of the complex. Erythrocyte invasion by the malaria merozoite is rapid, going to completion within seconds of the initial interaction between parasite and red cell surface (39). Over such a short time span, could the presence of blocking antibodies interfere with the ability of processing-inhibitory antibodies to bind the merozoite surface and prevent both processing and invasion? To address this question directly in an in vitro system, a series of invasion experiments was performed. Mature, biosynthetically radiolabeled T9/96 schizonts were washed and placed in culture with fresh red cells. Merozoite release and red cell invasion were then allowed to proceed in the presence or absence of mAbs 12.8 and 12.10, with or without the additional presence of affinity-purified anti-pME6 human antibodies. The overall efficiency of invasion was assessed by counting the number of new ring stage parasites formed over the course of the experiment; MSP-1 processing in individual samples was subsequently assessed by direct immunoprecipitation of MSP-133 from the culture supernatants using mAb X509 coupled to Sepharose. In preliminary dose–response experiments, a concentration of ⩾400 μg ml−1 of either mAb 12.10 (Fig. 7 A) or mAb 12.8 (data not shown) was sufficient to reduce the amount of MSP-133 release to a level of inhibition seen in the presence of 5 mM EGTA, a potent inhibitor of MSP-1 secondary processing (11). The results of a typical experiment (of a total of three independent experiments) investigating the effects of the anti-pME6 blocking antibodies on the activity of mAbs 12.8 and 12.10 are presented in Fig. 7 B. In isolation, mAbs 12.8 and 12.10 virtually abolished both invasion (Fig. 7 B, bottom) and MSP-133 release (Fig. 7 B, top). However, in the presence of equal concentrations of the anti-pME6 human antibodies, the effects of mAb 12.8, but not of 12.10, were completely reversed (Fig. 7 B, lanes 5 and 7). Neither the anti-pME6 antibodies alone nor mAb 89.1 alone had any effect on either processing or invasion (Fig. 7 B, lanes 3 and 8), and mAb 89.1 exhibited no blocking activity (Fig. 7 B, lanes 9 and 10). These results unambiguously demonstrate that, under conditions of active release of viable merozoites, mAbs 12.8 and 12.10 effectively prevent both MSP-1 processing and erythrocyte invasion, and this activity can be efficiently abrogated by the presence of human blocking antibodies.

Bottom Line: Most significantly, affinity-purified, naturally acquired human antibodies specific for epitopes within the NH2-terminal 83-kD domain of MSP-1 very effectively block the processing-inhibitory activity of the anti-MSP-119 mAb 12.8.Blocking antibodies therefore (a) are part of the human response to malarial infection; (b) can be induced by MSP-1 structures unrelated to the MSP-119 target of processing-inhibitory antibodies; and (c) have the potential to abolish protection mediated by anti-MSP-119 antibodies.Our results suggest that an effective MSP-119-based falciparum malaria vaccine should aim to induce an antibody response that prevents MSP-1 processing on the merozoite surface.

View Article: PubMed Central - PubMed

Affiliation: Division of Parasitology, National Institute for Medical Research, London, United Kingdom.

ABSTRACT
Merozoite surface protein-1 (MSP-1) of the human malaria parasite Plasmodium falciparum undergoes at least two endoproteolytic cleavage events during merozoite maturation and release, and erythrocyte invasion. We have previously demonstrated that mAbs which inhibit erythrocyte invasion and are specific for epitopes within a membrane-proximal, COOH-terminal domain of MSP-1 (MSP-119) prevent the critical secondary processing step which occurs on the surface of the extracellular merozoite at around the time of erythrocyte invasion. Certain other anti-MSP-119 mAbs, which themselves inhibit neither erythrocyte invasion nor MSP-1 secondary processing, block the processing-inhibitory activity of the first group of antibodies and are termed blocking antibodies. We have now directly quantitated antibody-mediated inhibition of MSP-1 secondary processing and invasion, and the effects on this of blocking antibodies. We show that blocking antibodies function by competing with the binding of processing-inhibitory antibodies to their epitopes on the merozoite. Polyclonal rabbit antibodies specific for certain MSP-1 sequences outside of MSP-119 also act as blocking antibodies. Most significantly, affinity-purified, naturally acquired human antibodies specific for epitopes within the NH2-terminal 83-kD domain of MSP-1 very effectively block the processing-inhibitory activity of the anti-MSP-119 mAb 12.8. The presence of these blocking antibodies also completely abrogates the inhibitory effect of mAb 12.8 on erythrocyte invasion by the parasite in vitro. Blocking antibodies therefore (a) are part of the human response to malarial infection; (b) can be induced by MSP-1 structures unrelated to the MSP-119 target of processing-inhibitory antibodies; and (c) have the potential to abolish protection mediated by anti-MSP-119 antibodies. Our results suggest that an effective MSP-119-based falciparum malaria vaccine should aim to induce an antibody response that prevents MSP-1 processing on the merozoite surface.

Show MeSH
Related in: MedlinePlus