Acute Plasmodium chabaudi chabaudi malaria infection induces antibodies which bind to the surfaces of parasitized erythrocytes and promote their phagocytosis by macrophages in vitro

Abstract

CBA/Ca mice infected with 5 x 10(4) Plasmodium chabaudi chabaudi AS-parasitized erythrocytes experience acute but self-limiting infections of relatively short duration. Parasitemia peaks ( approximately 40% infected erythrocytes) on day 10 or 11 and is then partially resolved over the ensuing 5 to 6 days, a period referred to as crisis. How humoral and cellular immune mechanisms contribute to parasite killing and/or clearance during crisis is controversial. Humoral immunity might be parasite variant, line, or species specific, while cellular immune responses would be relatively less specific. For P. c. chabaudi AS, parasite clearance is largely species and line specific during this time, which suggests a primary role for antibody activity. Accordingly, acute-phase plasma (APP; taken from P. c. chabaudi AS-infected mice at day 11 or 12 postinfection) was examined for the presence of parasite-specific antibody activity by enzyme-linked immunosorbent assay. Antibody binding to the surface of intact, live parasitized erythrocytes, particularly those containing mature (trophozoite and schizont) parasites, was demonstrated by immunofluorescence in APP and the immunoglobulin G (IgG)-containing fraction thereof. Unfractionated APP (from P. c. chabaudi AS-infected mice), as well as its IgG fraction, specifically mediated the opsonization and internalization of P. c. chabaudi AS-parasitized erythrocytes by macrophages in vitro. APP from another parasite line (P. c. chabaudi CB) did not mediate the same effect against P. c. chabaudi AS-parasitized erythrocytes. These results, which may represent one mechanism of parasite removal during crisis, are discussed in relation to the parasite variant, line, and species specificity of parasite clearance during this time.

FIG. 1

ELISA analysis of binding properties of antibody present during P. c. chabaudi AS infection. CBA/Ca mice were infected with 5 × 10⁴ PE, and the course of parasitemia was monitored (⧫). Plasma samples from the days indicated were collected and tested against PE antigen preparation, measured by ELISA for total Ig, IgM, IgG1, and IgG2a and expressed as the mean absorbance reading. Antibody levels were also measured in NP. All data are expressed as means of three independent experiments ± standard deviation.

FIG. 2

Immunofluorescence analysis of antibody in APP binding to the surface of PE and E. E (A and B) or PE containing either young parasites (C and D) or mature parasites (E and F) were incubated with NP (A, C, and E) or APP (B, D, and F), and antibody binding to the cell surface was quantified in duplicate samples by FACS analysis. The proportion of cells within the predetermined window of positive fluorescence is indicated. The window was determined by using E or PE incubated with KGS alone instead of a plasma sample.

FIG. 3

Kinetics of antibody activity during a P. c. chabaudi AS infection analyzed by surface immunofluorescence. Plasma samples from the days indicated were collected and tested against intact live PE in triplicate samples. Antibody binding to the PE surface was also measured in NP. The results represent the percentage of positive PE and were determined by using a window defined by PE incubated with KGS alone instead of plasma. All data are expressed as means of three independent experiments ± standard deviation.

FIG. 4

Immunofluorescence analysis of anti-P. c. chabaudi AS antibody subclasses from APP binding specifically to the surface of PE. PE containing mature parasites were incubated with NP or APP, and the antibody isotype binding to the cell surface was identified by immunofluorescence and quantified in triplicate samples by FACS analysis. The results represent the percentage of positive PE and were determined by using a window defined by PE incubated with KGS alone instead of plasma. All data are expressed as means of three independent experiments ± standard deviation.

FIG. 5

Immunofluorescence analysis of the line specificity of APP antibody. E (A, C, and E) or PE (B, D, and F) were incubated with NP (A and B), homologous anti-P. c. chabaudi AS APP (C and D), and heterologous anti-P. c. chabaudi CB APP (E and F), and antibody binding was detected in triplicate samples. The window was determined by using E or PE incubated with KGS alone instead of plasma.

FIG. 6

Phagocytosis of PE preincubated with homologous or heterologous APP (HOMOAPP or HETEROAPP). PE containing mature parasites, and E, were incubated with NP, APP, or KGS (BLK) and then exposed to macrophages in vitro. The results are presented as the phagocytic index (percentage of macrophages with PE inside) for the different treatments and are further broken down by the numbers of cells inside individual macrophages.

FIG. 7

Phagocytosis of PE preincubated with protein G-fractionated APP fractions. PE containing mature parasites were incubated with NP, APP, or their protein G fractions and then exposed to macrophages in vitro. The results are presented as the phagocytic index (percentage of macrophages with PE inside) for the different treatments. Analysis by t test showed no significant difference between the APP and the IgG fraction obtained from it.