Linkage group selection: towards identifying genes controlling strain specific protective immunity in malaria

Abstract

Protective immunity against blood infections of malaria is partly specific to the genotype, or strain, of the parasites. The target antigens of Strain Specific Protective Immunity are expected, therefore, to be antigenically and genetically distinct in different lines of parasite. Here we describe the use of a genetic approach, Linkage Group Selection, to locate the target(s) of Strain Specific Protective Immunity in the rodent malaria parasite Plasmodium chabaudi chabaudi. In a previous such analysis using the progeny of a genetic cross between P. c. chabaudi lines AS-pyr1 and CB, a location on P. c. chabaudi chromosome 8 containing the gene for merozoite surface protein-1, a known candidate antigen for Strain Specific Protective Immunity, was strongly selected. P. c. chabaudi apical membrane antigen-1, another candidate for Strain Specific Protective Immunity, could not have been evaluated in this cross as AS-pyr1 and CB are identical within the cell surface domain of this protein. Here we use Linkage Group Selection analysis of Strain Specific Protective Immunity in a cross between P. c. chabaudi lines CB-pyr10 and AJ, in which merozoite surface protein-1 and apical membrane antigen-1 are both genetically distinct. In this analysis strain specific immune selection acted strongly on the region of P. c. chabaudi chromosome 8 encoding merozoite surface protein-1 and, less strongly, on the P. c. chabaudi chromosome 9 region encoding apical membrane antigen-1. The evidence from these two independent studies indicates that Strain Specific Protective Immunity in P. c. chabaudi in mice is mainly determined by a narrow region of the P. c. chabaudi genome containing the gene for the P. c. chabaudi merozoite surface protein-1 protein. Other regions, including that containing the gene for P. c. chabaudi apical membrane antigen-1, may be more weakly associated with Strain Specific Protective Immunity in these parasites.

Figure 1. Mixed strain infections of Plasmodium chabaudi chabaudi CB and AJ in mice pre-immunised with either strain, or in a non-immune batch mate.

(A) shows total parasitaemias during the course of mixed strain infections in immunised and non-immune mice as measured by microscopic examination of thin blood smears stained with Giemsa's stain: non-immune mouse (dotted line with open circles), two CB-immunised mice (dashed lines with filled symbols), two AJ-immunised mice (unbroken lines with filled symbols). Strain specific parasitaemias (AJ in pink; CB in green) are shown (B) in the non-immune mouse, (C) in the two CB-immunised mice and (D) in the two AJ-immunised mice. The strain specific parasitaemias in the mixed strain infections were calculated from the total parasitaemias measured on thin blood smears stained with Giemsa's stain and from the proportions of CB and AJ parasites in the mixtures as determined using strain specific RTQ-PCR (see text). Squares and triangles represent mouse 1 and 2, respectively, in the CB and AJ immunised mice in Fig. 1A, C and D.

Figure 2. Course of blood stage induced infection of the uncloned CB x AJ cross progeny grown in a non-immune mouse (dotted line with open circles), in a CB-immunised mouse (pink line filled squares) and in an AJ-immunised mouse (blue line with filled symbols).

Arrows indicate day of infection when the uncloned cross progeny grown in the immunised mice or the non-immune mouse were sub-inoculated for expansion in non-immune mice (see text).

Figure 3. The Comparative Intensities of 92 AFLP markers of Plasmodium chabaudi chabaudi strain AJ from the progeny of a genetic cross between P. c. chabaudi strains CB-pyr10 and AJ following selection in mice immunised strain AJ (see text).

AJ-specific markers (indicated by black diamonds) were located on a P. c. chabaudi genetic linkage map, generated from a genetic cross between AS and AJ . Numbers after letter ‘C’ and ‘g’ represent P. c. chabaudi chromosome numbers and P. c. chabaudi unassigned linkage groups, respectively, in the genetic linkage map . Of the six AJ markers which were most reduced under AJ-specific immune selection (see Table 2), five (indicated by asterisks) could be located to P. c. chabaudi chromosome 8, forming a selection valley with the P. c. chabaudi msp-1 gene at its lowest point (see text). RTQ-PCR values for the proportions of the AJ-immune selected cross progeny carrying the AJ alleles of the Merozoite Surface Protein-1 (msp-1) are indicated by the red triangle and Apical Membrane Antigen-1 (ama-1) by the green triangle in the AJ-immune selected cross progeny. The red line indicates Comparative Intensity of 50%.