Unit: Biomedical Parasitology
Director: Pierre Druilhe
The focus of the work performed at the Biomedical Parasitology Unit is on immune interactions between human beings and Plasmodium falciparum, both at the level of pre-erythrocytic stages (the sporozoite and the liver stage)and that of the merozoite stage of the erythrocytic cycle. Studies in humans are aimed at relating immune responses with resistance to infection so as to provide a rationale for the development of vaccines. Results of the first MSP-3 blood stage vaccine show long term memory with persistence, one year after immunisation, of strong immune responses and the biological activity against the parasite of the antibodies elicited in humans. A phase-I.b study has been performed in 2003 in Burkina Faso and has confirmed the safety of the formulation. Three formulations of the liver stage antigen LSA-3 are now about to enter clinical trials. Meanwhile, improved laboratory models have been developed that provide tools to analyse immune effector mechanisms and identify antigens relevant to protection.
I - Immunity to erythrocytic stages
We have chosen an approach where clinical observations, interactions between P. falciparum and humans and material derived from these studies, are at the origin of the paradigms and the research orientations. By passively transferring protection by African immune IgG into naive infected subjects, we identified a mechanism of defence where antibodies cooperate with monocytes, called ADCI. The latter was used to screen a P. falciparum genomic library to identify an antigen, called MSP-3 and to identify the critical role of cytophilic antibodies, IgG1 and IgG3, able to cooperate with monocytes. Studies conducted this year are inkeeping with this bio-clinical approach of an antimalarial vaccine, they were directed at collecting further evidence of the role of MSP-3 , conducting a clinical trial with it, identifying in detail the epitopes targeted by protective antibodies, and identifying further targets of the same defence mechanism.
The results of the first clinical trial performed with MSP-3 had provided last year extremely valuable information that could not be gathered in experimental models. Besides safety and immunogenicity, it was shown that the Ab's elicited in humans were able to fully block the erythrocytic cycle of Plasmodium falciparum in a monocyte dependent manner. This study has been complemented this year by the analysis of serum samples collected one year after immunisation. Results showed at one year high T-cell responses, particularly in the group receiving the antigen adjuvated with alum, and the persistence of Ab's with high affinity. Above all, the Ab's collected one year post-immunisation were still able to to kill Plasmodium falciparum blood stages, both under in vitro conditions in the monocyte dependent ADCI assay, as well as under in vivo conditions by passive transfer into P. falciparum infected immuno-compromised SCID mice. Thus, the MSP-3 vaccine trial showed that low doses of MSP-3 injected with simple adjuvants, such as alum, readily induced Ab's of cytophilic classes, directed to fully conserved epitopes, very long lasting and with strong biological activity against Plasmodium falciparum erythrocytic stages.
We have furthered our analysis of the MSP-3 multi-gene family, particularly with respect to gene expression, protein expression on merozoite surface, antigenicity, biological activity of the corresponding antibody, cross-reactivity between homologous epitopes within the family, T-cell responses (performed in Senegal), and sequence conservation in various Plasmodium falciparum isolates. Finally, another series of 6 peptides has been used to complement the epitope mapping performed within the MSP-3-1 antigen with MSP-3-2 (or MSP-6) antigen. Results show that this multi-gene family differs in many respects from other Plasmodium falciparum multi-gene families described so far and their characterisation suggest that they play an important role in eliciting immune responses involved in parasite-density control and, in general, in defence mechanisms in the human host. In total, the gene family shows highly conserved divergences between genes, with high degree of homologies between cross-reactive epitopes. The results have fundamental implications in the natural host / parasite interaction and practical consequences for vaccine development.
A phase-I.b study of the original MSP-3 LSP formulation was undertaken at the CNRLP in Ouagadougou, Burkina Faso, which was supported by the African Malaria Network and received a technical assistance from the European Malaria Vaccine Initiative. This study, performed in 30 African adults, confirms the excellent safety of the alum adjuvated formulation in individuals already primed by the parasite in endemic areas.
Numerous progresses were made in the analysis of the monocyte dependent mode of action of antimalarial Ab's. The analysis focused on the characteristics of the antigen triggering the MN-dependent antiparasitic effect, the differentiation status of the monocytes in order to obtain a parasitostatic effect, the receptors implicated at the monocyte surface level, the physico-chemical characteristics of the monokines involved in the effect upon surrounding intra-erythrocytic parasites and the respective role of IgG1 as compared to IgG3 antibodies.
We have also furthered our analysis of the interaction between worms and malaria in 5 different settings, and confirmed the strong deleterious effect of worm carriage upon the occurrence of malaria attacks. Moreover, immunological studies have been performed in one of those settings and have provided illuminating information about the immunological effect of worm carriage upon the quality of the antibody response to protective malarial antigens, thereby bringing new information about the reasons for the long delay in the establishment of a strong immune protection against malaria under field conditions.
We have showed that the P. falciparum immuno-compromised mouse erythrocytic stages model was a valuable one for the assessment of novel antimalarial compounds (studies performed with GSK). These studies also contribute to a better understanding of the means of controlling non-adaptive defences and thereby to develop an improved mouse model that could be more easily used by other research groups.
II - pre-erythrocytic stages
We have shown that what has long been considered an " anti-sporozoite immunity " is in fact related to the development of a liver-phase trophozoite and have focused therefore our efforts on the study of immunity to liver stages. The lead molecule LSA-3 has been identified by differential screening of immune responses from protected vs. non-protected human volunteers, both immunised by irradiated sporozoites. We had, last year, characterised an additional 29 new pre-erythrocytic genes and have this year developed a method providing access to the transcriptome of this poorly known stage. The first achievement has been the elucidation of the P.yoelii liver stage transcriptome.
We had recently characterised a novel liver stage antigen, LSA-5, and obtained evidence, by various means, of an essential role in protection of this new pre-erythrocytic antigen, particularly by immunisation and challenge of aotus monkeys. Further work has been performed with this molecule and particularly immuno-epidemiological studies have revealed an extremely strong association between immune responses to LSA-5 and protection acquired against malaria under field conditions. This result strengthens very much the importance of this new molecule which is now going to be evaluated in clinical trials and evaluated in pre-clinical screens in combination with LSA-3, for its potential to provide, improved protection against P. falciparum sporozoite challenge.
We capitalised on the recent availability of Plasmodium and mouse genomic data, the development of high throughput sequencing which, associated with optimally infected cultures, led us to elaborate a strategy for the analysis of the hepatic stage transcriptome and provided a proof of concept for the feasibility of the approach which can be now applied also to P. falciparum. RTPCR data with some of the novel genes provided comparative data on the transcriptional patterns of these genes in the various forms of the parasite found in the mosquito and in the vertebrate host. The majority of the transcribed genes identified encode for predicted proteins of as yet unknown function.
We have employed the technique recently developed to analyse in situ intra-hepatic cellular responses in the liver and compared them with peripheral blood or splenocyte responses. The results of in situ investigations provide data on intra-hepatic epitope specific T-cell recruitment and shows major differences with responses measured in peripheral blood. These experiments thus provide a rationale for the design of antigen presentation methods that elicit immune responses that are stronger where they are needed, around the intra-hepatic parasites.
We have recently obtained the development of P. falciparum liver stages in human hepatocytes grafted in an improved immuno-compromised mouse model. This novel mouse model, developed in collaboration with the group of Christian Brechot, was obtained by capitalising on knowledge acquired with the grafting of P. falciparum erythrocytic stages. We combined two genetic deficiencies affecting mouse hepatocytes and mouse T- and B-cell lymphocytes, with a pharmacological treatment aimed at controlling non-adaptative defences. Results show that human hepatocytes can survive in medium to long term in this novel model and keep their fully differentiated functions : secretion of serum protein by human hepatocytes remains unchanged over time, and the injection of P. falciparum sporozoites by intra-venous route in grafted mice resulted in invasion of human hepatocytes and the development of fully mature liver forms, i.e. twice as large as that developing in vitro. Given the extremely scarce access to human hepatocytes, the development of this novel mouse model for P. falciparum liver stages provides an essential tool for fundamental studies on host / parasite interactions, applied studies for the development of drugs and vaccines, as well as potentially a model for formal genetic studies (genetic crosses).
Finally, the GMP grade preparation of 3 formulations derived from the LSA-3 antigen, which had been delayed for administrative and technical reasons, is now again on its way and should be tested in phase-I and II trial in human volunteers in the coming year.
Keywords: Plasmodium falciparum, immunity, vaccines, merozoite, hepatic stages, gene expression