Sylviane PIED

Immunologie Fondamentale et Clinique des Maladies Parasitaires

The BCIP team results from the fusion of the “Malaria Immunopathophysiology” group 1 headed by Sylviane Pied from the Infectious Immunophysiopathology Unit, CNRS URA 1961, Department of Immunology at the Institut Pasteur in Paris and the “Clinical Parasite Immunology” group 2 headed by Gilles Riveau working on schistosomiasis and malaria in Africa and Emmanuel Herman, both from Inserm U 547, Institut Pasteur de Lille. The team 1 has joined the Institut Pasteur of Lille in January 2008 and was supported by the “Programme d’Incitation à la Mobilité d’Equipe” PIME) of CNRS and Inserm U 547.

1. Malaria Immunophysiopathology

The malaria parasite life cycle involves 2 obligatory hosts, and infection results from intricate interactions between the parasite and the mosquito, and then man. Because these 2 hosts are separated by hundreds of millions of years of divergent evolution, successful parasite survival is contingent on its own evolutionary adaptation to both of them. To adapt, the parasite must use host signals and even host immune responses to achieve parasitism. The immune responses of the vertebrate host during malaria infection are complex and not well understood, although different animal models provide insight into possible effector mechanisms. In the light of the complexity of Plasmodium development, different immune response components evidently operate at different stages of its life cycle in different organs, but little is known about their fine specificity, dynamics, regulation and/or influence on infection outcome. While some of these responses are protective, others help the parasite to evade those measures or induce pathology. The infection induces the interaction of overlapping complex responses and signals of the immune system, including T-cells and cytokine/chemokine networks, which may play a central role in the outcome of the disease. The diversity of the responses induced may depend on the complex composition of the parasite (including antigenic, superantigenic and mitogenic activities) that may lead to an overstimulation of the immune system. Using experimental models, C57BL/6 (B6) mice infected with either Plasmodium (P.) yoelii (protective responses) or P. berghei ANKA (pathological responses) and cohorts of P. falciparum infected patients, we analyzed lymphocyte populations involved in the different immune responses to determine their phenotype as well as their repertoire diversity, their dynamic and their function at physiological state and during pathogenesis.

1.1  Immune responses involved in protection against primary P.yoelii infection.

Most studies exploring protective immune responses during Plasmodium infection focused on reactions to immunization with irradiated sporozoites or candidate vaccines. Little is known about the wide variety of responses induced by primary infection. In addition, our understanding of protective immunity remains incomplete. We used the experimental model of  B6 mice infected with P. yoelii 265 By. The mice develop parasitemia that is rapidly and spontaneously cured. The advantage of using the P. yoelii model is that infection can be initiated by injecting either sporozoites, to obtain a complete cycle of plasmodium within the mammalian host (intrahepatic schizonts and blood stages) or by injecting blood stages. Thus, this system enables differentiate between responses specific to liver stages or blood stages. In this model, we analysed the immune response both at cellular and at molecular levels. We determined the kinetics and phenotypes of responding T cells and analyzed their functions during infection. Our studies mainly focus on the intrahepatic T cells. We demonstrated these last years that non-conventional natural killer ab (NKT) cells, a major T lymphocyte population in the liver that recognizes glycolipids presented by a non–classical MHC class I molecule, CD1d, highly conserved among species, are implicated in the first defense mechanisms against P. yoelii liver stage. The NKT cells elicited in P. yoelii-infected B6 mouse livers are heterogeneous and composed of CD1d-dependent invariant CD4+ NKT cells and of CD4CD8 double-negative (DN) NKT cells, which include CD1d-dependent invariant and non-invariant cells, and CD1d-independent cells (Soulard et al., 2007). During infection, parasite-activated hepatic DN NKT cells secrete interferon (IFN)g and tumor necrosis factor (TNF)a (Th1 cytokines). These cells isolated from infected mice inhibited intra-hepatic development of the parasite in vitro in a CD1d-dependent manner. Pertinently, the parasite burden was higher in the livers of B6.CD1d-deficient (no NKT cells) than in those of B6 mice demonstrating thus that NKT cells and CD1d may be involved in the early control of primary P. yoelii infection.

Clearly we demonstrate that innate immune responses mediated by NK and NKT cells participate in early immune mechanisms that control Plasmodium infection mainly by acting on Plasmodium pre-erythrocytic stages (Roland, 2006; Soulard, 2007). One of the common factors of the P. yoelii induced NK and NKT cells response is their release of IFNγ upon in vivo stimulation during infection. Consequently, we examined the kinetics of pro-inflammatory cytokines such as IFNγ, TNFα and IL-12 production during P. yoelii sporozoites-induced infection in B6 mice. We found that the early immune response to sporozoite-induced malaria is characterize by a peak of IFNγ in the serum at  day 5 of infection produced by splenic CD4 T cells. We also observed that IFNγ deficient mice develop less parasitemia than wild type. Altogether these results challenge the current view regarding the role of IFNγ in the immune response to Plasmodium. An early IFNγ production during malaria can be deleterious for the host. They data also highlight the complex regulation of the primary immune response to malaria (Soulard, 2009).

1.2  Immune responses associated with CM pathology induced by P. berghei ANKA.

Cerebral Malaria (CM) occurs in 10-15% of P. falciparum infected patients and is responsible for high mortality. CM is characterized by 2 main factors: sequestration of parasitized erythrocytes and the T-cell response. To date, the exact mechanisms causing CM have not yet been clearly established, but compelling evidence implicates T lymphocytes in its development. Animal models, even if they do not reproduce all the features of human CM, enabled us to directly demonstrate T-cell involvement. Our findings linked CM development to abT cells in mice infected with P. berghei ANKA, a strain able to induce CM in susceptible mice. Thus, we characterized the abT-cells subsets and studied their pathogenic functions. We demonstrated that CM development in P. berghei ANKA -infected mice was correlated with a significantly higher number of CD8+TCRVβ8+ cells in peripheral blood (PBL) and in the brains of mice that had been infected with sporozoites or blood stage parasites than mice not developing CM (CM). Phenotypically, these CD8+ T cells were activated and they secreted IFNγ and TNFα, two cytokines known to be implicated in CM. In spite of all these indications, the exact role played by these CD8+ T cells is still unknown. In addition, the mechanisms involved in the control of the balance between the efficient immune responses that control parasite infection and inefficient responses leading to chronic infections or pathological manifestations are unknown. As a result, we consider that one of the mechanisms could be an inefficient control of P. berghei ANKA-induced pathogenic effector T cells by regulatory T cells (Treg). Therefore, we evaluated the role of Treg cells in CM in P. berghei ANKA infected B6 mice. We found more activated CD4+CD25high T cells expressing Foxp3, with a biased CDR3 TCRVß repertoire and in vitro suppressive function during the course of the infection. However, in vivo those cells do not protect against CM, because CM was not exacerbated in mice depleted of their CD25+ Treg cells. Moreover, this absence of protection was not due to in vivo blockade of the Treg cells suppressive function by interleukin (IL)-6 produced during the infection, because IL-6–depleted or –deficient B6 mice were as susceptible as the controls to CM (Vigário AM.; 2007).

1.3  Identification of genetic traits controlling host resistance to CM.

Host genes are among the main determinants of CM susceptibility/resistance. Initial studies conducted using 12 inbred mouse strains (derived from ancestor pairs trapped in different localities in Europe and North Africa) led to the identification of five strains highly resistant to CM, whereas most laboratory mouse strains are susceptible. Using a genome-wide screening approach, we were able to link the phenotype of CM resistance to marker loci located in two different chromosomal regions Berr 1 (chromosome 1) and Berr 2 (chromosome 11), using a backcross cohort derived from the CM resistant strains (WLA) and the C57BL/6 laboratory strain (CM susceptible). On the other hand, the analysis of F2 mice derived from the same combination (WLA and C57BL/6) revealed three disease phenotypes. Two of them, CMSHPS (B6 phenotype) and CMRHPS (WLA phenotype) were expected, and a third new phenotype, CMRHPR, emerged. The animals displaying the latter are able to control their parasitemia efficiently. This resistance phenotype has not been reported previously and offers a promising experimental model of chronic malaria that appears more relevant to the human disease than the animal models currently used. We have also obtained evidence for linkage of this HP resistance phenotype to Berr 1 and to another marker locus on chromosome 9 (Berr 3). Congenic mouse strains for the chromosomal regions so far identified respectively associated with CM and HP resistance are under construction.

1.4  Studies on Pf-infected patients.


To elucidate the role of immune response in the pathogenesis of P. falciparum infection and particularly in CM, a study was started on 2 cohorts of patients from different geographic areas: 1) Gondia, India, an endemic village in northeastern Maharashtra state where transmission is seasonal (collaboration Dr. Gyan C. Mishra, NCCS, Pune, India, project funded by IFCPAR) and 2) Libreville, Gabon, where transmission is holo-endemic (collaboration Pr. Maryvonne Kombila, Department of Parasitology-Mycology, Libreville Hospital Center, Gabon, PAL+ project funded). The Gabonese cohorts are constituted of 310 children (140 girls and 170 boys) of 2 months to 5 years-old. The Indian cohorts consist of approximately 100 adult patients, including endemic and non–endemic uninfected controls. P. falciparum infected individuals were divided into five groups according to World Health Organization criteria: 1) asymptomatic (AM), 2) uncomplicated malaria (UM), 3) severe non-cerebral malaria (SNCM), 4) CM  and 5) individuals that recovered from CM (ex CM). After obtaining informed consent (patients or their parents or guardians) and Ethics Committee approval, blood samples were collected at hospital admission (day 0), then on days 7 and 30 after treatment.

Due to the multi-factorial character of malaria, we used an integrated approach that addresses numerous interconnected aspects of transmission, infection, immune responses and disease by studying 1) T cell populations looking at their phenotypes and repertoire diversity; 2) global profiles of autoreactive antibodies to brain proteins produced during infection using the PANAMA-BLOT method, 3) Profile of P. falciparum specific antibodies, 4) P. falciparum genotypings and 5) patterns of circulating cytokines.

Several observations have already been made from these studies. In particular, a correlation between an increase of self-reacting antibodies with disease severity. It is well estabkshed that hypergammaglobulinemia and polyclonal B-cell activation commonly occur in malaria, a fraction of antibodies produced recognize self-components from various tissues and organs. However, it is unclear whether these autoantibodies play a role in protective immunity or contribute to the events leading to development of neurological complications. We found that CM patients develop a high autoantibody response to two brain autoantigens, the alpha-2-spectrin (Guiyedi, 2007) and the beta-tubulin-3 (Bansal, 2009). These autoantibody reactivities were correlated positively with increased plasma concentrations of cytokines that have been previously associated with CM. These data strongly suggest the involvement of an autoimmune counterpart targeting brain antigens in CM. Clearly, the role of autoreactive antibodies in the pathogenesis of CM, and other severe malaria manifestations should be investigated further.

In conclusion, this multiparametric analytical approach, combined with multivariate statistical analyses (MANOVA, discriminant analysis, k-mean clustering) should help us to elucidate the role of the immune response in the outcome of P. falciparum infection. We hope that this strategy will allow us to define the signatures of the various components of the immune response associated with clinical subphenotypes of P. falciparum malaria.



2. Clinical Immunology of Malaria and Schistosomiasis

Since 1992, our group has developed two different approaches on immunity against schistosomiasis, i) laboratory research devoted to vaccine development using the schistosome 28-kDa glutathione S-transferase (28GST), and ii) studies on human acquired immunity in the endemic region of the Senegal River. Since the region, like numerous sub-saharian regions, is endemic for malaria, research in the field has been extended to studies on acquired immunity during co-infection (schistosomiasis and malaria). Today, our research is oriented to the clinical development of our schistosomiasis vaccine candidate, and to various environmental factors influencing the immune response against malaria infection and its pathology, including co-infection, nutritional environment and vector density.


2.1 Immuno-pharmacology of vaccine

Vector design for mucosal or cutaneous vaccination against schistosomiasis has been extensively developed. Synthetic (liposomes, microparticules) and recombinant live vectors (BCG, attenuated B. pertussis, attenuated S. thyphi) were engineered in collaboration with several European laboratories including the C. Locht team. All these vectors were adapted for mucosal immunisation and provided a unique panel of tools and models to develop future vaccine processes. As an example, a single-dose mucosal administration with biodegradable microparticles produced by spray-drying technique with particular polyester polymers and entrapping Schistosoma mansoni antigen, was enough to generate protective immune response in a mouse model. A nucleic acid vaccine was also developed using the 28GST gene. Beside the fact that this kind of vaccine presentation could be very effective in experimental models, we determined that the constitution of the DNA vector is a crucial element, which could influence the quality of the immune response and could be adapted to generate appropriate effects. We showed that a methylated CpG-containing plasmid has a particular role in the orientation and the amplitude of the induced immune response, and this action was effective when these specific motifs were functionally recognized by Toll-like receptor 9 (TLR9).


2.2 Immuno-epidemiological studies on schistosomiasis and malaria

We studied acquired immunological reactions during schistosomiasis in patients from endemic areas. These epidemiological studies have been completed by a programme focusing on the changes in the immune responses during co-infection, associating schistosomiasis and malaria. In this way, we started to develop research on the causes of variability of the host response during parasite infection and their consequences on pathogenesis. These studies shed light on the immune response acquired during natural infection and helped us to design protocols of clinical trials adapted to the selected target population. Recently, in collaboration with UR024 (F. Simondon & F. Remoué) and UR016 (D. Fontenille) of IRD, the laboratory of vector and parasite ecology of Dakar University (UCAD; L. Konaté), the medical entomology laboratory of Pasteur Institute of Dakar (I. Dia), and the parasite biology and epidemiology laboratory of IMTSSA (Ch. Rogier, Marseille), we developed immunological studies in the field on the relationships between the human host and vectors in the context of malaria infections. We determined the influence of environmental heterogeneities on vector bionomics and malaria epidemiology in the Senegal River basin, and the variability of the antibody response to P. falciparum in children according to their exposure of Anopheles gambiae s.l or Anopheles funestus vectors.


2.3  Clinical development of schistosomiasis vaccine

Progress has been made to limit the disease severity by using chemotherapy, but continuous re-infection and risks of drug resistance point to the necessary development of alternative strategies. It is widely agreed that immunological prevention of chronic parasitic infections will be extremely difficult to achieve. Conversely, in some major helminth infections like schistosomiasis, where parasite egg laying in the tissues is the exclusive cause of pathology, inhibition of parasite fecundity might represent for the future a novel way to prevent the deleterious effects of these chronic infections in man. The concept to target by vaccination the cause of the pathology (fecundity) rather than the parasite itself has been developed by our laboratory. Our work has designated 28GST as a potent candidate for therapeutic vaccination, and should provide a potent tool to control a major chronic infection. In the frame of the clinical development of our vaccine candidate against schistosomiasis, the 28-kDa GST of S. haematobium (Sh28GST), the team initiated, developed and coordinated several clinical studies in humans. This protein, selected as a therapeutic vaccine candidate, has been produced under GMP conditions by Eurogentec S.A., and has been evaluated in pre-clinical toxicological studies. Following a first safety phase undertaken in the CIC of Lille and funded by Inserm, three clinical phases in Senegal and one in Niger demonstrated that this candidate was safe and immunogenic in infected children, which represent the target population. These clinical studies were supported by an EC demonstration project.

We will pursue the projects that have shown the most potential and promise. Basic and field studies will be conducted in parallel, often in a highly interactive manner to study the additive role of environment and host-parasite interactions in malaria and in its degrees of severity. We will 1) evaluate the appropriateness of the immune response during malaria, 2) identify biomarkers associated to cerebral malaria, 3) identify host genetic factors associated with protection or pathological processes and 4) characterize environmental factors that modulate these responses.

Understanding the duality of immune responses to malaria is expected to contribute to the design of new therapeutics and vaccine strategies.

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