The Unit's long term objective is to develop vaccines against Plasmodium blood stages. Four research teams work on complementary research programmes, including a vaccine development programme, studies on field parasite diversity and an integrated, multifaceted analysis of the host/parasite interactions contributing to malaria pathology. One of the characteristics of the Unit is to privilege in vivo studies in humans and in experimental monkey models.
As a consequence, we have a strong involvement in collaborative studies in the field: molecular epidemiology studies in Senegal and French Guiana with the Réseau International des Instituts Pasteur; hospital-based studies on clinical malaria in Ghana; studies on experimental monkey infections in French Guiana and Sri Lanka.
1. The vaccine development programme focuses on P. falciparum and P. vivax asexual blood stage antigens identified as targets of immune protective mechanisms.
MSP-1: (S.Longacre, I. Holm, A. Manamperi). S. Longacre's group develops a programme targeting the conserved C-terminus of the merozoite surface antigen MSP-1 using baculovirus-expressed recombinant proteins. The natural P. cynomolgi / toque monkey system is used as a primate model. Vaccination trials carried out in this model have provided solid evidence for indicating that the MSP-1p19 antigen is a potent protective antigen with a relatively long lasting effect against homologous and heterologous strains. Advances in the vaccine research programme on P. falciparum MSP-1p19 are such that applications for phase I trials with MSP-1 p19 are currently under review.
In parallel, the analysis of the immune response to the C-terminal conserved and polymorphic domains of P. vivax MSP-1have been conducted. Finally, the crystalline structure of the P. falciparum MSP-1p19 has been determined in collaboration with the Unité d'mmunologie Structurale. It confirms the existence in this molecule of the two EGF domains which had been observed in the crystalline structure of the P.cynomolgi MSP-1p19.
R23 (O.Puijalon, P.Dubois, M. Huynh Quan Dat) : The R23 antigen is a target of antibodies which opsonize P.falciparum-infected erythrocytes. Vaccination experiments in squirrel monkeys have indicated that protection could be induced after immunization with a minimal dose of R23 repeats. The antibody response to R23 in humans exposed to P.falciparum was explored in a holo and a mesoendemic setting in Senegal. Seroprevalence and antibody dynamics were similar in both settings; the rainy season was associated with a reduced prevalence in children suggesting absorption of antibodies onto circulating parasites.
2. Studies on field parasite diversity and its consequences on immune responses ( O. Puijalon, H. Jouin, D. Schleiermacher )
Comparative genotyping of peripheral blood and placental parasites in Senegalese women showed that in 50/58 cases, the genotype profiles were only partially concordant, with alleles observed both in peripheral blood and in the placenta, together with other alleles observed in one territory only. This non random genotype distribution within the host indicates organ-specific sequestration and raises serious concern on how best monitoring P. falciparum infections in pregnant women during pregnancy and, in more general terms, on how accurately examination of peripheral blood of an infected patient reflects any P. falciparum infection.
The allele-specific humoral response to the highly polymorphic P. falciparum MSP-1 block 2 has been studied in villagers from two Senegalese villages, Dielmo (perennial transmission) and N'Diop (seasonal transmission), taking advantage of the rich longitudinal series of blood samples along with highly documented clinical and parasitological data. In Dielmo, longitudinal follow-up over several years outlined a remarkably fixed response, with identical reactivity profiles, independent of the past or current parasite types, and identical light chain and heavy chain distribution over the years. These results indicate that there is no accumulation of novel specificities with exposure to an increasingly large number of alleles. This unpredicted pattern, highly reminiscent of clonal imprinting, strongly contrasts with the progressive accumulation of specificities described for the variant antigen PfEMP1. As they stand, our results suggest that eliciting such an imprinted response is yet another type of immune evasion mechanism developed by the malaria parasites, favouring an inappropriately fitted response to novel alleles and types.
Parasite diversity was comparatively analysed in different clinical groups in French Guyana, a hypoendemic region where malaria occurs in isolated foci. Transmission is low and peripheral health system is efficiently organized to provide prompt treatment and thus reduce prolonged carriage. This all leads to remarkably limited parasite polymorphisme and a large proportion of single genotype infections. Such a setting with a limited parasite gene pool and non immune subjects provides a unique opportunity to investigate the consequences of parasite polymorphism on disease severity. Analysis of msp1, msp2 , glurp and var repertoire genotypes in patients with mild or severe malaria showed that severe malaria was associated with infection by a specific genotype association. Thus, the long supposed increased virulence of some strains has found here its first field demonstration.
3. Parasite pathogenicity factors (S. Bonnefoy, P. Pendyala, M. Diez Silva, M. Guillotte, P. David, O. Puijalon).
We have initiated a reverse genetics approach to investigate the consequences of gene knockout or allelic replacement of specific parasite factors on the in vivo infection patterns, as well as a systematic transcriptome analysis under different physiological conditions.
Comparative genotype analysis of different parasite strains in the spelnectomized Saimiri monkey had indicated that strain pathogenicity was associated with a total resa deletion and a specific hrp1 allele. We have thus invalidated the resa gene of the FUP/CB parasites, a moderately virulent strain for the Saimiri monkey. Analysis of the properties of the red blood cell membrane are underway, in particular comparative biophysical assays in which rigidity and strength of adhesion are measured. Analysis of the in vivo consequences of the resa invalidation of the FUP/CP parasites on parasite multiplication rate and associated pathological signs in the Saimiri monkey is commencing.
We have pursued the development of novel reverse genetic tools, including development of novel selectable markers and construction of GFP-tagged parasites.
Our first objective in using DNA array technology has been to explore the modulation of parasite transcription by the spleen. Indeed, the spleen is known to influence red cell surface expression/exposure of parasite antigens. Differential screening of DNA microarrays using a mung-bean digested genomic DNA shotgun microarray identified 85 and 100 clones over-expressed in animals with and without a spleen, respectively. This include genes coding for polypeptides linked to cytoadherence and/or antigenic variation. It is particularly interesting to note that this analysis has pointed out modulation of the expression of the conserved knob accessory molecules that are under current investigation as potential virulence factors.
4. Contribution of immune activation to malaria pathology. (C.Behr, S. Loizon, P.Boeuf, F.Remerand , J.C.Michel).
We have carried out an analysis of the activation of gdT lymphocytes in African children, including a detailed repertoire analysis in healthy children and in children suffering from a malaria attack. This has shown that the peripheral representation of the various gdT cell subsets in healthy African subjects (children and adults) differs from what is described in Europeans. In children with acute malaria, the Vd1 subset is transiently activated. Vg chain analysis did not show any preferential chain association. Immunoscope analysis did not outline any preferential clonal expansion. In these children, Vg9 T cells are also activated, but with a smaller extent. Ex-vivo intracellular staining showed that the Vg9 T cells are high producer of TNF-a while Vd1 are in comparison high producer of IFN-g. Overall numbers of circulating gd cells and the Vd1/ Vg9 ratio did not significantly differ in the various clinical groups. The results obtained so far are compatible with the proposed role for the Vd1 population in the regulation of the immune system and the return to homeostasis.
Further analysis of the cytokine profiles associated with distinct forms of clinical malaria showed that cerebral malaria is associated with a statistically significant higher level of IL-2R than severe anaemia or uncomplicated malaria. Severe anaemia is associated with lower levels of TNFR (I and II).
Sepsis and severe sepsis present an interesting analogy with mild and severe malaria, with systemic inflammation as a major hallmark of pathology. In collaboration with the Intensive Care Unit of the Hopital Lariboisière, Paris, we conducted a longitudinal follow up of patients suffering severe sepsis or local intense inflammation. We measured monocyte surface HLA-DR and co-stimulatory molecules CD80 and CD54 by quantitative Flow cytometry either ex-vivo or after in vitro LPS stimulation. This showed that "monocyte deactivation" is a general phenomenon that induces down regulation of HLA-DR, but also of other co-stimulatory molecules that play a major role in T cell activation.
We have devoted considerable effort to establishing the experimental conditions and the immunological and genetic tools necessary for physiopathology studies in the squirrel monkey. Monoclonal antibodies have been screened towards functional identification of mononuclear cell subsets. This has allowed us to establish the normal range of cellular subset and activation profiles in non infected naive animals as well as to analyse the kinetics of activation of the various subsets during P.falciparum infection.