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.

A single parasite gene determines strain-specific protective immunity against malaria: the role of the merozoite surface protein I.

Despite many decades of research, no registered vaccine against the pathogenic blood stages of the malaria parasite exists, translating into the loss of many hundreds of thousands of young lives each year in tropical Africa. Although many parasite proteins have been shown to induce immune responses in the host, proof for their induction of protective immunity is still lacking. We previously reported a novel genetic approach called linkage group selection (LGS) for rapid identification of target antigens of strain-specific protective immunity (SSPI) against malaria. In preliminary LGS experiments, we crossed two genetically distinct strains of Plasmodium chabaudi chabaudi and subjected their progeny to selection in strain-specifically immunised mice, measuring the effects of SSPI selection with low coverage/resolution genetic markers. In the present study, through application of high coverage/resolution, single nucleotide polymorphism (SNP) markers spanning all 14 parasite chromosomes, we analysed 35 SSPI selection events on different populations of progeny parasites. Here we report a comprehensive high resolution genome-wide analysis of the effects of strain-specific immune selection on blood stage parasites. Our analyses consistently identify a single genomic region spanning approximately 79kb on chromosome 8 as the region controlling SSPI. Within this region, one gene (that of merozoite surface protein 1, MSP-1) accounted for >60% of genetic polymorphism and was most frequently under greatest reduction under SSPI. These results, combined with those of an independent LGS analysis of a different genetic cross with different parental strains, demonstrate that more than any other locus, the gene for MSP-1 determines the effect of strain-specific protective immunity against malaria in these host-parasite combinations. Our results provide unique insight into the precise timing of the parasite killing immune response against progeny parasites carrying specific alleles of MSP-1; these findings pave the way for investigating which part(s) of this highly polymorphic molecule mediate the protective immune response.

Gene encoding erythrocyte binding ligand linked to blood stage multiplication rate phenotype in Plasmodium yoelii yoelii.

Variation in the multiplication rate of blood stage malaria parasites is often positively correlated with the severity of the disease they cause. The rodent malaria parasite Plasmodium yoelii yoelii has strains with marked differences in multiplication rate and pathogenicity in the blood. We have used genetic analysis by linkage group selection (LGS) to identify genes that determine differences in multiplication rate. Genetic crosses were generated between genetically unrelated, fast- (17XYM) and slowly multiplying (33XC) clones of P. y. yoelii. The uncloned progenies of these crosses were placed under multiplication rate selection in blood infections in mice. The selected progenies were screened for reduction in intensity of quantitative genetic markers of the slowly multiplying parent. A small number of strongly selected markers formed a linkage group on P. y. yoelii chromosome 13. Of these, that most strongly selected marked the gene encoding the P. yoelii erythrocyte binding ligand (pyebl), which has been independently identified by Otsuki and colleagues [Otsuki H, et al. (2009) Proc Natl Acad Sci USA 106:10.1073/pnas.0811313106] as a major determinant of virulence in these parasites. In an analysis of a previous genetic cross in P. y. yoelii, pyebl alleles of fast- and slowly multiplying parents segregated with the fast and slow multiplication rate phenotype in the cloned recombinant progeny, implying the involvement of the pyebl locus in determining the multiplication rate. Our genome-wide LGS analysis also indicated effects of at least 1 other locus on multiplication rate, as did the findings of Otsuki and colleagues on virulence in P. y. yoelii.

Strain-specific immunity may drive adaptive polymorphism in the merozoite surface protein 1 of the rodent malaria parasite Plasmodium chabaudi.

Clinical immunity against malaria is slow to develop, poorly understood and strongly strain-specific. Understanding how strain-specific immunity develops and identifying the parasite antigens involved is crucial to developing effective vaccines against the disease. In previous experiments we have shown that strain-specific protective immunity (SSPI) exists between genetically distinct strains (cloned lines) of the rodent malaria parasite Plasmodium chabaudi chabaudi in mice [Cheesman, S., Raza, A., Carter, R., 2006. Mixed strain infections and strain-specific protective immunity in the rodent malaria parasite P. chabaudi chabaudi in mice. Infect. Immun. 74, 2996-3001]. In two subsequent studies, we identified the highly polymorphic Merozoite Surface Protein 1 (MSP-1) as being the principal candidate molecule for the control of SSPI against P. c. chabaudi malaria [Martinelli et al., 2005; Pattaradilokrat, S., Cheesman, S.J., Carter R., 2007. Linkage group selection: towards identifying genes controlling strain-specific protective immunity in malaria. PLoS ONE 2(9):e857]. In the present study, we sequenced the whole msp1 gene of several genetically distinct strains of P. chabaudi and found high levels of genetic diversity. Protein sequence alignments reveal extensive allelic polymorphism between the P. chabaudi strains, concentrated primarily within five regions of the protein. The 3'-end sequence region, encoding the C-terminal 21 kDa region (MSP-1(21)), which is analogous and homologous to MSP-1(19) of Plasmodium falciparum, appears to have been subject to balancing selection. We have found that the strains with the lowest sequence identity at MSP-1(21) (i.e. AS/CB and AJ/CB) induce robust and reciprocal SSPI in experimental mice. In contrast, two strains that do not induce reciprocal SSPI are identical at the 21 kDa region. Final identification of the region(s) controlling SSPI will provide important information to help guide decisions about MSP-1 based vaccines.

Congenicity and genetic polymorphism in cloned lines derived from a single isolate of a rodent malaria parasite.

Many of the most commonly studied lines of the rodent malaria parasite Plasmodium yoelii yoelii originated from a single parasite isolate designated 17X. Amongst these lines, however, are parasites that exhibit variation in genotype and phenotype (e.g. growth rate). We describe here the results of a comparative genetic analysis between cloned lines of 17X that differ in growth rate, using nucleotide sequences of specific genes and patterns of genome-wide amplified fragment length polymorphism (AFLP). Our findings indicate that the original stock of 17X comprises two unrelated genotypes. Genotype-1 is represented by parasites with a slow growth phenotype (e.g. 17X (NIMR)) and a fast growth phenotype (e.g. 17XYM). Within this genotype, there are also genomic differences manifest as a small number of AFLP bands that differentiate the fast- and slow-growing lines from each other. The other genotype, genotype-2, is represented only by parasites with a slow growth phenotype (e.g. 17XA).

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.

Linkage Group Selection--a fast approach to the genetic analysis of malaria parasites.

Genetic analysis of malaria parasites has shown that the mechanisms of inheritance in these organisms are classically Mendelian. In other words, alleles of genes at different loci recombine, and alleles at the same gene locus segregate, in the progeny of a genetic cross between two genetically distinct lines of malaria parasite. Importantly, such progeny are haploid in the first filial generation following genetic crossing. Consequently, genetic analysis, including linkage analysis, can be done directly upon the cloned cross progeny. Linkage analysis conducted upon the progeny of genetic crosses between malaria parasites can lead to the location of a single gene controlling a specific phenotype, as has been achieved to identify the gene for chloroquine resistance in Plasmodium falciparum. The work involved, however, is extremely labour intensive. It involves the generation of many hundreds, to a thousand or so, of independent recombinant clones from the cross progeny and the biological characterisation, and genetic typing for hundreds of molecular genetic markers of each such clone. We discuss here a fast-track method for identifying genes controlling specific phenotypes, e.g. drug resistance/sensitivity. It involves the mass screening with quantitative molecular genetic markers of the uncloned progeny of a genetic cross following its growth under a selection pressure representing the phenotype of interest. We have called the method Linkage Group Selection.

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.

Virulence and competitive ability in genetically diverse malaria infections.

Explaining parasite virulence is a great challenge for evolutionary biology. Intuitively, parasites that depend on their hosts for their survival should be benign to their hosts, yet many parasites cause harm. One explanation for this is that within-host competition favors virulence, with more virulent strains having a competitive advantage in genetically diverse infections. This idea, which is well supported in theory, remains untested empirically. Here we provide evidence that within-host competition does indeed select for high parasite virulence. We examine the rodent malaria Plasmodium chabaudi in laboratory mice, a parasite-host system in which virulence can be easily monitored and competing strains quantified by using strain-specific real-time PCR. As predicted, we found a strong relationship between parasite virulence and competitive ability, so that more virulent strains have a competitive advantage in mixed-strain infections. In transmission experiments, we found that the strain composition of the parasite populations in mosquitoes was directly correlated with the composition of the blood-stage parasite population. Thus, the outcome of within-host competition determined relative transmission success. Our results imply that within-host competition is a major factor driving the evolution of virulence and can explain why many parasites harm their hosts.

A genetic approach to the de novo identification of targets of strain-specific immunity in malaria parasites.

Vaccine research in malaria has a high priority. However, identification of specific antigens as candidates for vaccines against asexual blood stages of malaria parasites has been based on largely circumstantial evidence. We describe here how genes encoding target antigens of strain-specific immunity in malaria can be directly located in the parasite's genome without prior information concerning their identity, by the method we call linkage group selection. Two genetically distinct clones of the rodent malaria parasite Plasmodium chabaudi chabaudi, each of which induces an immunity in laboratory mice that is more protective against challenge with itself than with the heterologous strain, were genetically crossed, and the uncloned cross progeny selected in mice that had been made partially immune by infection and drug cure with one or the other parental strain. Proportions of parental alleles in the selected and unselected cross progeny were compared by using quantitative genome-wide molecular markers. A small number, including groups of linked markers forming so-called selection valleys, were markedly reduced under strain-specific immune pressure. A very prominent selection valley was found to contain the gene for merozoite surface protein-1, a major candidate antigen for malaria vaccine development, at the locus at which the strongest reduction under strain-specific immune selection was detected. Closely linked to the merozoite surface protein-1 gene was a gene containing the signature motif of the ring-infected erythrocyte surface antigen family. Another affected locus, unlinked to this selection valley, contained a member of the serine repeat antigen gene family.

Amplified fragment length polymorphism measures proportions of malaria parasites carrying specific alleles in complex genetic mixtures.

We are interested in developing a method for the identification of those genes in malaria parasites which underlie a variety of selectable phenotypes of the parasites including drug resistance and strain-specific immunity. A key aspect of our approach is to subject a genetically mixed population of malaria parasites to a specific phenotypic selection pressure such as the administration of an antimalarial drug and then identify genetic markers affected by the selection. Our aim, therefore, is to be able to identify those genetic markers carried by sensitive parasites which disappear from the population after selection as they should be closely linked to the locus determining the phenotype involved. We have previously identified more than 800 amplified fragment length polymorphisms (AFLP) distinguishing two cloned strains of the rodent malaria parasite Plasmodium chabaudi chabaudi and distributed across the whole of the parasites' genome. Here we evaluate the possibility that the intensities of these AFLP bands are quantitatively related to the proportions of parasite DNA which bear these markers in mixtures of genetically different parasites. We prepared mixtures of DNA and parasitised blood from different mixtures of two genetically distinct clones (AS and AJ) of P. c. chabaudi and analysed AFLP markers amplified from them. The results show that the relative band intensities of AFLP markers are, indeed, linearly related to the proportions of parasite DNA in a genetically mixed sample. The precision of the method is sufficient to detect reliably the effects of phenotypic selection at loci closely linked to a genetic locus under selection.

Host heterogeneity is a determinant of competitive exclusion or coexistence in genetically diverse malaria infections.

During an infection, malaria parasites compete for limited amounts of food and enemy-free space. Competition affects parasite growth rate, transmission and virulence, and is thus important for parasite evolution. Much evolutionary theory assumes that virulent clones outgrow avirulent ones, favouring the evolution of higher virulence. We infected laboratory mice with a mixture of two Plasmodium chabaudi clones: one virulent, the other avirulent. Using real-time quantitative PCR to track the two parasite clones over the course of the infection, we found that the virulent clone overgrew the avirulent clone. However, host genotype had a major effect on the outcome of competition. In a relatively resistant mouse genotype (C57B1/6J), the avirulent clone was suppressed below detectable levels after 10 days, and apparently lost from the infection. By contrast, in more susceptible mice (CBA/Ca), the avirulent clone was initially suppressed, but it persisted, and during the chronic phase of infection it did better than it did in single infections. Thus, the qualitative outcome of competition depended on host genotype. We suggest that these differences may be explained by different immune responses in the two mouse strains. Host genotype and resistance could therefore play a key role in the outcome of within-host competition between parasite clones and in the evolution of parasite virulence.

Real-time quantitative PCR for analysis of genetically mixed infections of malaria parasites: technique validation and applications.

A technique that can distinguish and quantify genetically different malaria parasite clones in a mixed infection reliably and with speed and accuracy would be very useful for researchers. Many current methods of genotyping and quantification fall down on a number of aspects relating to their ease of use, sensitivity, cost, reproducibility and, not least, accuracy. Here we report the development and validation of a method that offers several advantages in terms of cost, speed and accuracy over conventional PCR or antibody-based methods. Using real-time quantitative PCR (RTQ-PCR) with allele-specific primers, we have accurately quantified the relative proportions of clones present in laboratory prepared ring-stage mixtures of two genetically distinct clones of the rodent malaria parasite Plasmodium chabaudi chabaudi. Accurate and reproducible measurement of the amount of genomic DNA representing each clone in a mixture was achieved over 100-fold range, corresponding to 0.074% parasitised erythrocytes at the lower end. To demonstrate the potential utility of this method, we include an example of the type of application it could be used for. In this case, we studied the growth rate dynamics of mixed-clone infections of P. chabaudi using an avirulent/virulent clone combination (AS (PYR) and AJ) or two clones with similar growth rate profiles (AQ and AJ). The modification of the technique described here should enable researchers to quickly extract accurate and reliable data from in-depth studies covering broad areas of interest, such as analyses of clone-specific responses to drugs, vaccines or other selection pressures in malaria or other parasite species that also contain highly polymorphic DNA sequences.