Linkage Group Selection: Towards Identifying Genes Controlling Strain Specific Protective Immunity in Malaria

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

Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites§

Artemisinin- and artesunate-resistant Plasmodium chabaudi mutants, AS-ART and AS-ATN, were previously selected from chloroquine-resistant clones AS-30CQ and AS-15CQ respectively. Now, a genetic cross between AS-ART and the artemisinin-sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS-30CQ and AS-ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS-ATN to artemisinin derivates, the other cannot account solely for the resistance of AS-ART, relative to the responses of its sensitive progenitor AS-30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug-response phenotype was not genetically stable. No mutations in the UBP-1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed

Mixed Strain Infections and Strain-Specific Protective Immunity in the Rodent Malaria Parasite Plasmodium chabaudi chabaudi in Mice

Important to malaria vaccine design is the phenomenon of “strain-specific” immunity. Using an accurate and sensitive assay of parasite genotype, real-time quantitative PCR, we have investigated protective immunity against mixed infections of genetically distinct cloned “strains” of the rodent malaria parasite Plasmodium chabaudi chabaudi in mice. Four strains of P. c. chabaudi, AS, AJ, AQ, and CB, were studied. One round of blood infection and drug cure with a single strain resulted in a partial reduction in parasitemia, compared with levels for naïve mice, in challenge infections with mixed inocula of the immunizing (homologous) strain and a heterologous strain. In all cases, the numbers of blood-stage parasites of each genotype were reduced to similar degrees. After a second, homologous round of infection and drug cure followed by challenge with homologous and heterologous strains, the parasitemias were reduced even further. In these circumstances, moreover, the homologous strain was reduced much faster than the heterologous strain in all of the combinations tested. That the immunity induced by a single infection did not show “strain specificity,” while the immunity following a second, homologous infection did, suggests that the “strain-specific” component of protective immunity in malaria may be dependent upon immune memory. The results show that strong, protective immunity induced by and effective against malaria parasites from a single parasite species has a significant “strain-specific” component and that this immunity operates differentially against genetically distinct parasites within the same infection

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).

<p>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).</p

Physical and genetic locations of AFLP markers of <i>Plasmodium chabaudi chabaudi</i> strain AJ whose Comparative Intensities (CI) were reduced below 50% in the progeny of the genetic cross between CB-pyr10 and AJ following selection in an AJ-immunised mouse (see text).

<p>The six AJ markers with CI of <20% in the AJ-immune selected cross progeny mapped to positions closely linked to the gene encoding the <i>P. c. chabaudi</i> merozoite surface protein-1 (MSP-1) are indicated in bold. ND not determined.</p>*<p>The first and second numbers in brackets represent percentages of parasite DNA carrying the AJ alleles of the indicated gene ( <i>msp</i>-1 or <i>ama</i>-1), respectively in the AJ-immune selected cross progeny and in the non-immune selected cross progeny, as measured by RTQ-PCR (see text)</p>**<p>Numbers after ‘<i>pf</i>’’ indicate the <i>Plasmodium falciparum</i> chromosome number followed by distance along the chromosome in kilo base pairs</p

The parameters used to calculate the predicted maximum number of recombinant lines present in the pooled progeny of the genetic cross between strains CB-pyr10 and AJ of <i>Plasmodium chabaudi chabaudi</i>.

<p>The predicted number of such recombinants is calculated as described (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000857#s4" target="_blank"><i>Materials and Methods</i></a>). SEM, standard error of mean.</p

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

<p>AJ-specific markers (indicated by black diamonds) were located on a <i>P. c. chabaudi</i> genetic linkage map, generated from a genetic cross between AS and AJ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000857#pone.0000857-Martinelli2" target="_blank">[34]</a>. Numbers after letter ‘C’ and ‘g’ represent <i>P. c. chabaudi</i> chromosome numbers and <i>P. c. chabaudi</i> unassigned linkage groups, respectively, in the genetic linkage map <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000857#pone.0000857-Martinelli2" target="_blank">[34]</a>. Of the six AJ markers which were most reduced under AJ-specific immune selection (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000857#pone-0000857-t002" target="_blank">Table 2</a>), five (indicated by asterisks) could be located to <i>P. c. chabaudi</i> chromosome 8, forming a selection valley with the <i>P. c. chabaudi msp</i>-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 (<i>msp</i>-1) are indicated by the red triangle and Apical Membrane Antigen-1 (<i>ama</i>-1) by the green triangle in the AJ-immune selected cross progeny. The red line indicates Comparative Intensity of 50%.</p

Strain-specific protective effect of the immunity induced by live malarial sporozoites under chloroquine cover.

The efficacy of a whole-sporozoite malaria vaccine would partly be determined by the strain-specificity of the protective responses against malarial sporozoites and liver-stage parasites. Evidence from previous reports were inconsistent, where some studies have shown that the protective immunity induced by irradiated or live sporozoites in rodents or humans were cross-protective and in others strain-specific. In the present work, we have studied the strain-specificity of live sporozoite-induced immunity using two genetically and immunologically different strains of Plasmodium cynomolgi, Pc746 and PcCeylon, in toque monkeys. Two groups of monkeys were immunized against live sporozoites of either the Pc746 (n = 5), or the PcCeylon (n = 4) strain, by the bites of 2-4 sporozoite-infected Anopheles tessellates mosquitoes per monkey under concurrent treatments with chloroquine and primaquine to abrogate detectable blood infections. Subsequently, a group of non-immunized monkeys (n = 4), and the two groups of immunized monkeys were challenged with a mixture of sporozoites of the two strains by the bites of 2-5 infective mosquitoes from each strain per monkey. In order to determine the strain-specificity of the protective immunity, the proportions of parasites of the two strains in the challenge infections were quantified using an allele quantification assay, Pyrosequencing™, based on a single nucleotide polymorphism (SNP) in the parasites' circumsporozoite protein gene. The Pyrosequencing™ data showed that a significant reduction of parasites of the immunizing strain in each group of strain-specifically immunized monkeys had occurred, indicating a stronger killing effect on parasites of the immunizing strain. Thus, the protective immunity developed following a single, live sporozoite/chloroquine immunization, acted specifically against the immunizing strain and was, therefore, strain-specific. As our experiment does not allow us to determine the parasite stage at which the strain-specific protective immunity is directed, it is possible that the target of this immunity could be either the pre-erythrocytic stage, or the blood-stage, or both