Genomic and transcriptomic comparisons of closely related malaria parasites differing in virulence and sequestration pattern.

Background: Malaria parasite species differ greatly in the harm they do to humans. While P. falciparum kills hundreds of thousands per year, P. vivax kills much less often and P. malariae is relatively benign. Strains of the rodent malaria parasite Plasmodium chabaudi show phenotypic variation in virulence during infections of laboratory mice. This make it an excellent species to study genes which may be responsible for this trait. By understanding the mechanisms which underlie differences in virulence we can learn how parasites adapt to their hosts and how we might prevent disease. Methods: Here we present a complete reference genome sequence for a more virulent P. chabaudi strain, PcCB, and perform a detailed comparison with the genome of the less virulent PcAS strain. Results: We found the greatest variation in the subtelomeric regions, in particular amongst the sequences of the pir gene family, which has been associated with virulence and establishment of chronic infection. Despite substantial variation at the sequence level, the repertoire of these genes has been largely maintained, highlighting the requirement for functional conservation as well as diversification in host-parasite interactions. However, a subset of pir genes, previously associated with increased virulence, were more highly expressed in PcCB, suggesting a role for this gene family in virulence differences between strains. We found that core genes involved in red blood cell invasion have been under positive selection and that the more virulent strain has a greater preference for reticulocytes, which has elsewhere been associated with increased virulence. Conclusions: These results provide the basis for a mechanistic understanding of the phenotypic differences between Plasmodium chabaudi strains, which might ultimately be translated into a better understanding of malaria parasites affecting humans.

Plasmodium-specific atypical memory B cells are short-lived activated B cells.

A subset of atypical memory B cells accumulates in malaria and several infections, autoimmune disorders and aging in both humans and mice. It has been suggested these cells are exhausted long-lived memory B cells, and their accumulation may contribute to poor acquisition of long-lasting immunity to certain chronic infections, such as malaria and HIV. Here, we generated an immunoglobulin heavy chain knock-in mouse with a BCR that recognizes MSP1 of the rodent malaria parasite, Plasmodium chabaudi. In combination with a mosquito-initiated P. chabaudi infection, we show that Plasmodium-specific atypical memory B cells are short-lived and disappear upon natural resolution of chronic infection. These cells show features of activation, proliferation, DNA replication, and plasmablasts. Our data demonstrate that Plasmodium-specific atypical memory B cells are not a subset of long-lived memory B cells, but rather short-lived activated cells, and part of a physiologic ongoing B-cell response.

Follicular Helper T Cells are Essential for the Elimination of Plasmodium Infection.

CD4+ follicular helper T (Tfh) cells have been shown to be critical for the activation of germinal center (GC) B-cell responses. Similar to other infections, Plasmodium infection activates both GC as well as non-GC B cell responses. Here, we sought to explore whether Tfh cells and GC B cells are required to eliminate a Plasmodium infection. A CD4 T cell-targeted deletion of the gene that encodes Bcl6, the master transcription factor for the Tfh program, resulted in complete disruption of the Tfh response to Plasmodium chabaudi in C57BL/6 mice and consequent disruption of GC responses and IgG responses and the inability to eliminate the otherwise self-resolving chronic P. chabaudi infection. On the other hand, and contrary to previous observations in immunization and viral infection models, Signaling Lymphocyte Activation Molecule (SLAM)-Associated Protein (SAP)-deficient mice were able to activate Tfh cells, GC B cells, and IgG responses to the parasite. This study demonstrates the critical role for Tfh cells in controlling this systemic infection, and highlights differences in the signals required to activate GC B cell responses to this complex parasite compared with those of protein immunizations and viral infections. Therefore, these data are highly pertinent for designing malaria vaccines able to activate broadly protective B-cell responses.

ICAM-1 is a key receptor mediating cytoadherence and pathology in the Plasmodium chabaudi malaria model.

BACKGROUND: Parasite cytoadherence within the microvasculature of tissues and organs of infected individuals is implicated in the pathogenesis of several malaria syndromes. Multiple host receptors may mediate sequestration. The identity of the host receptor(s), or the parasite ligand(s) responsible for sequestration of Plasmodium species other than Plasmodium falciparum is largely unknown. The rodent malaria parasites may be useful to model interactions of parasite species, which lack the var genes with their respective hosts, as other multigene families are shared between the species. The role of the endothelial receptors ICAM-1 and CD36 in cytoadherence and in the development of pathology was investigated in a Plasmodium chabaudi infection in C57BL/6 mice lacking these receptors. The schizont membrane-associated cytoadherence (SMAC) protein of Plasmodium berghei has been shown to exhibit reduced CD36-associated cytoadherence in P. berghei ANKA-infected mice. METHODS: Parasite tissue sequestration and the development of acute stage pathology in P. chabaudi infections of mice lacking CD36 or ICAM-1, their respective wild type controls, and in infections with mutant P. chabaudi parasites lacking the smac gene were compared. Peripheral blood parasitaemia, red blood cell numbers and weight change were monitored throughout the courses of infection. Imaging of bioluminescent parasites in isolated tissues (spleen, lungs, liver, kidney and gut) was used to measure tissue parasite load. RESULTS: This study shows that neither the lack of CD36 nor the deletion of the smac gene from P. chabaudi significantly impacted on acute-stage pathology or parasite sequestration. By contrast, in the absence of ICAM-1, infected animals experience less anaemia and weight loss, reduced parasite accumulation in both spleen and liver and higher peripheral blood parasitaemia during acute stage malaria. The reduction in parasite tissue sequestration in infections of ICAM-1 null mice is maintained after mosquito transmission. CONCLUSIONS: These results indicate that ICAM-1-mediated cytoadherence is important in the P. chabaudi model of malaria and suggest that for rodent malarias, as for P. falciparum, there may be multiple host and parasite molecules involved in sequestration.

Antibody-independent mechanisms regulate the establishment of chronic Plasmodium infection.

Malaria is caused by parasites of the genus Plasmodium. All human-infecting Plasmodium species can establish long-lasting chronic infections1-5, creating an infectious reservoir to sustain transmission1,6. It is widely accepted that the maintenance of chronic infection involves evasion of adaptive immunity by antigenic variation7. However, genes involved in this process have been identified in only two of five human-infecting species: Plasmodium falciparum and Plasmodium knowlesi. Furthermore, little is understood about the early events in the establishment of chronic infection in these species. Using a rodent model we demonstrate that from the infecting population, only a minority of parasites, expressing one of several clusters of virulence-associated pir genes, establishes a chronic infection. This process occurs in different species of parasites and in different hosts. Establishment of chronicity is independent of adaptive immunity and therefore different from the mechanism proposed for maintenance of chronic P. falciparum infections7-9. Furthermore, we show that the proportions of parasites expressing different types of pir genes regulate the time taken to establish a chronic infection. Because pir genes are common to most, if not all, species of Plasmodium10, this process may be a common way of regulating the establishment of chronic infections.

Blood-stage immunity to Plasmodium chabaudi malaria following chemoprophylaxis and sporozoite immunization.

Protection against malaria in humans can be achieved by repeated exposure to infected mosquito bites during prophylactic chloroquine treatment (chemoprophylaxis and sporozoites (CPS)). We established a new mouse model of CPS immunization to investigate the stage and strain-specificity of malaria immunity. Immunization with Plasmodium chabaudi by mosquito bite under chloroquine cover does not generate pre-erythrocytic immunity, which is acquired only after immunization with high sporozoite doses. Instead, CPS immunization by bite elicits long-lived protection against blood-stage parasites. Blood-stage immunity is effective against a virulent, genetically distinct strain of P. chabaudi. Importantly, if exposure to blood-stage parasitemia is extended, blood-stage parasites induce cross-stage immunity targeting pre-erythrocytic stages. We therefore show that CPS immunization can induce robust, long-lived heterologous blood-stage immunity, in addition to protection against pre-erythrocytic parasites following high dose sporozoite immunization. Cross-stage immunity elicited by blood-stage parasites may further enhance efficacy of this immunization regimen.