Anne Charlotte Grüner

Practical PCR genotyping protocols for Plasmodium vivax using Pvcs and Pvmsp1

BackgroundPlasmodium vivax is the second most prevalent malaria parasite affecting more than 75 million people each year, mostly in South America and Asia. In addition to major morbidity this parasite is associated with relapses and a reduction in birthweight. The emergence and spread of drug resistance in Plasmodium falciparum is a major factor in the resurgence of this parasite. P. vivax resistance to drugs has more recently emerged and monitoring the situation would be helped, as for P. falciparum, by molecular methods that can be used to characterize parasites in field studies and drug efficacy trials.MethodsPractical PCR genotyping protocols based on polymorphic loci present in two P. vivax genetic markers, Pvcs and Pvmsp1, were developed. The methodology was evaluated using 100 P. vivax isolates collected in Thailand.Results and DiscussionAnalysis revealed that P. vivax populations in Thailand are highly diverse genetically, with mixed genotype infections found in 26 % of the samples (average multiplicity of infection = 1.29). A large number of distinguishable alleles were found for the two markers, 23 for Pvcs and 36 for Pvmsp1. These were generally randomly distributed amongst the isolates. A total of 68 distinct genotypes could be enumerated in the 74 isolates with a multiplicity of infection of 1.ConclusionThese results indicate that the genotyping protocols presented can be useful in the assessment of in vivo drug efficacy clinical trials conducted in endemic areas and for epidemiological studies of P. vivax infections.

Invasion of host cells by malaria parasites: a tale of two protein families

Malaria parasites are obligate intracellular parasites whose invasive stages select and invade the unique host cell in which they can develop with exquisite specificity and efficacy. Most studies aimed at elucidating the molecules and the mechanisms implicated in the selection and invasion processes have been conducted on the merozoite, the stage that invades erythrocytes to perpetuate the pathological cycles of parasite multiplication in the blood. Bioinformatic analysis has helped identify the members of two parasite protein families, the reticulocyte‐binding protein homologues (RBL) and erythrocyte binding like (EBL), in recently sequenced genomes of different Plasmodium species. In this article we review data from classical studies and gene disruption experiments that are helping to illuminate the role of these proteins in the selection‐invasion processes. The manner in which subsets of proteins from each of the families act in concert suggests a model to explain the ability of the parasites to use alternate pathways of invasion. Future perspectives and implications are discussed.

Control of pathogenic CD8+ T cell migration to the brain by IFN-γ during experimental cerebral malaria

Previous studies have shown that IFN‐γ is essential for the pathogenesis of cerebral malaria (CM) induced by Plasmodium berghei ANKA (PbA) in mice. However, the exact role of IFN‐γ in the pathway (s) leading to CM has not yet been described. Here, we used 129P2Sv/ev mice which develop CM between 7 and 14 days post‐infection with PbA. In this strain, both CD4+ and CD8+ T cells were involved in the effector phase of CM. When 129P2Sv/ev mice deficient in the IFN‐γ receptor α chain (IFN‐γR1) were infected with PbA, CM did not occur. Migration of leucocytes to the brain at the time of CM was observed in wild type (WT) but not in deficient mice. However, in the latter, there was an accumulation of T cells in the lungs. Analysis of chemokines and their receptors in WT and in deficient mice revealed a complex, organ‐specific pattern of expression. Up‐regulation of RANTES/CCL5, IP‐10/CCL3 and CCR2 was associated with leucocyte migration to the brain and increased expression of MCP‐1/CCL2, IP‐10/CCL3 and CCR5 with leucocyte migration to the lung. This shows that IFN‐γ controls trafficking of pathogenic T cells in the brain, thus providing an explanation for the organ‐specific pathology induced by PbA infection.

Sterile Protection against Malaria Is Independent of Immune Responses to the Circumsporozoite Protein

Background Research aimed at developing vaccines against infectious diseases generally seeks to induce robust immune responses to immunodominant antigens. This approach has led to a number of efficient bacterial and viral vaccines, but it has yet to do so for parasitic pathogens. For malaria, a disease of global importance due to infection by Plasmodium protozoa, immunization with radiation-attenuated sporozoites uniquely leads to long lasting sterile immunity against infection. The circumsporozoite protein (CSP), an important component of the sporozoites surface, remains the leading candidate antigen for vaccines targeting the parasites pre-erythrocytic stages. Difficulties in developing CSP-based vaccines that reproduce the levels of protection afforded by radiation-attenuated sporozoites have led us to question the role of CSP in the acquisition of sterile immunity. We have used a parasite transgenic for the CSP because it allowed us to test whether a major immunodominant Plasmodium antigen is indeed needed for the induction of sterile protective immunity against infection. Methodology/Main Findings We employed a P. berghei parasite line that expresses a heterologous CSP from P. falciparum in order to assess the role of the CSP in the protection conferred by vaccination with radiation-attenuated P. berghei parasites. Our data demonstrated that sterile immunity could be obtained despite the absence of immune responses specific to the CSP expressed by the parasite used for challenge. Conclusions We conclude that other pre-erythrocytic parasite antigens, possibly hitherto uncharacterised, can be targeted to induce sterile immunity against malaria. From a broader perspective, our results raise the question as to whether immunodominant parasite antigens should be the favoured targets for vaccine development.

Vaccination with Live Plasmodium yoelii Blood Stage Parasites under Chloroquine Cover Induces Cross-Stage Immunity against Malaria Liver Stage

Immunity to malaria has long been thought to be stage-specific. In this study we show that immunization of BALB/c mice with live erythrocytes infected with nonlethal strains of Plasmodium yoelii under curative chloroquine cover conferred protection not only against challenge by blood stage parasites but also against sporozoite challenge. This cross-stage protection was dose-dependent and long lasting. CD4+ and CD8+ T cells inhibited malaria liver but not blood stage. Their effect was mediated partially by IFN-γ, and was completely dependent of NO. Abs against both pre-erythrocytic and blood parasites were elicited and were essential for protection against blood stage and liver stage parasites. Our results suggest that Ags shared by liver and blood stage parasites can be the foundation for a malaria vaccine that would provide effective protection against both pre-erythrocytic and erythrocytic asexual parasites found in the mammalian host.

Recombinant Human IFN-α Inhibits Cerebral Malaria and Reduces Parasite Burden in Mice

Most C57BL/6 mice infected i.p. with Plasmodium berghei ANKA (PbA) die between 7 and 14 days with neurologic signs, and the remainder die later (>15 days) with severe anemia. Daily i.p. injections of a recombinant human IFN-α (active on mouse cells) prevented death by cerebral malaria (87% deaths in the control mice vs 6% in IFN-α-treated mice). The mechanisms of this IFN-α protective effect were multiple. IFN-α-treated, PbA-infected mice showed 1) a marked decrease in the number of PbA parasites in the blood mediated by IFN-γ, 2) less sequestered parasites in cerebral vessels, 3) reduced up-regulation of ICAM-1 expression in brain endothelial cells, 4) milder rise of blood levels of TNF, 5) increased levels of IFN-γ in the blood resulting from an increased production by splenic CD8+ T cells, and 6) fewer leukocytes (especially CD8+ T cells) sequestered in cerebral vessels. On the other hand, IFN-α treatment did not affect the marked anemia observed in PbA-infected mice. Survival time in IFN-α-treated mice was further increased by performing three blood transfusions over consecutive days.

Cerebral malaria: mysteries at the blood-brain barrier.

Cerebral malaria is the most severe pathology caused by the malaria parasite, Plasmodium falciparum. The pathogenic mechanisms leading to cerebral malaria are still poorly defined as studies have been hampered by limited accessibility to human tissues. Nevertheless, histopathology of post-mortem human tissues and mouse models of cerebral malaria have indicated involvement of the blood-brain barrier in cerebral malaria. In contrast to viruses and bacteria, malaria parasites do not infiltrate and infect the brain parenchyma. Instead, rupture of the blood-brain barrier occurs and may lead to hemorrhages resulting in neurological alterations. Here, we review the most recent findings from human studies and mouse models on the interactions of malaria parasites and the blood-brain barrier, shedding light on the pathogenesis of cerebral malaria, which may provide directions for possible interventions.

A role for immune responses against non-CS components in the cross-species protection induced by immunization with irradiated malaria sporozoites.

Immunization with irradiated Plasmodium sporozoites induces sterile immunity in rodents, monkeys and humans. The major surface component of the sporozoite the circumsporozoite protein (CS) long considered as the antigen predominantly responsible for this immunity, thus remains the leading candidate antigen for vaccines targeting the parasites pre-erythrocytic (PE) stages. However, this role for CS was questioned when we recently showed that immunization with irradiated sporozoites (IrrSpz) of a P. berghei line whose endogenous CS was replaced by that of P. falciparum still conferred sterile protection against challenge with wild type P. berghei sporozoites. In order to investigate the involvement of CS in the cross-species protection recently observed between the two rodent parasites P. berghei and P. yoelii, we adopted our gene replacement approach for the P. yoelii CS and exploited the ability to conduct reciprocal challenges. Overall, we found that immunization led to sterile immunity irrespective of the origin of the CS in the immunizing or challenge sporozoites. However, for some combinations, immune responses to CS contributed to the acquisition of protective immunity and were dependent on the immunizing IrrSpz dose. Nonetheless, when data from all the cross-species immunization/challenges were considered, the immune responses directed against non-CS parasite antigens shared by the two parasite species played a major role in the sterile protection induced by immunization with IrrSpz. This opens the perspective to develop a single vaccine formulation that could protect against multiple parasite species.

Are Extensive T Cell Epitope Polymorphisms in the Plasmodium falciparum Circumsporozoite Antigen, a Leading Sporozoite Vaccine Candidate, Selected by Immune Pressure?

Protective cellular immune responses depend on MHC presentation of pathogen-derived Ag fragments. MHC diversity renders this process sensitive to point mutations coding for altered amino acid sequence of the short target Ag-derived peptides epitopes. Thus, in a given host, a pathogen with an altered epitope sequence will be more likely to escape detection and elimination by the immune system. At a population level, selection by immune pressure will increase the likelihood of polymorphism in important pathogen antigenic epitopes. This mechanism of immune evasion is found in viruses and other pathogens. The detection of polymorphic hot spots in an Ag is often taken as a strong indication of its role in protective immunity. We provide evidence that polymorphisms in the T cell epitopes of a malaria vaccine candidate are unlikely to have been selected by immune pressure in the human host.

Expression of the Erythrocyte-Binding Antigen 175 in Sporozoites and in Liver Stages of Plasmodium falciparum

Screening of a Plasmodium falciparum genomic expression library for antigens expressed at the pre-erythrocytic stages resulted in the isolation of a recombinant phage (DG249) whose insert corresponded to regions II and III of a 175-kDa erythrocyte-binding antigen (EBA-175). EBA-175 is a parasite ligand implicated in red blood cell invasion. Reverse-transcriptase polymerase chain reaction, indirect immunofluorescent antibody test, and Western blot analysis confirmed that EBA-175 is expressed not only in blood-stage parasites but also in infected hepatocytes and on the sporozoite surface. The presence of EBA-175 on pre-erythrocytic parasites enhances the vaccine potential of this antigen by adding another target to the immune responses elicited by immunization.