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 parasite's 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

Inhibitory Effect of TNF-α on Malaria Pre-Erythrocytic Stage Development: Influence of Host Hepatocyte/Parasite Combinations

BACKGROUND: The liver stages of malaria parasites are inhibited by cytokines such as interferon-gamma or Interleukin (IL)-6. Binding of these cytokines to their receptors at the surface of the infected hepatocytes leads to the production of nitric oxide (NO) and radical oxygen intermediates (ROI), which kill hepatic parasites. However, conflicting results were obtained with TNF-alpha possibly because of differences in the models used. We have reassessed the role of TNF-alpha in the different cellular systems used to study the Plasmodium pre-erythrocytic stages. METHODS AND FINDINGS: Human or mouse TNF-alpha were tested against human and rodent malaria parasites grown in vitro in human or rodent primary hepatocytes, or in hepatoma cell lines. Our data demonstrated that TNF-alpha treatment prevents the development of malaria pre-erythrocytic stages. This inhibitory effect however varies with the infecting parasite species and with the nature and origin of the cytokine and hepatocytes. Inhibition was only observed for all parasite species tested when hepatocytes were pre-incubated 24 or 48 hrs before infection and activity was directed only against early hepatic parasite. We further showed that TNF-alpha inhibition was mediated by a soluble factor present in the supernatant of TNF-alpha stimulated hepatocytes but it was not related to NO or ROI. Treatment TNF-alpha prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified. CONCLUSIONS: Treatment TNF-alpha prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified. However, the nature of the cytokine-host cell-parasite combination must be carefully considered for extrapolation to the human infection

Événements immunologiques et physiologiques au stade pré-érythrocytaire de Plasmodium

Le cycle biologique de Plasmodium chez son hôte vertébré comprend deux stades : le stade pré-érythrocytaire qui englobe les sporozoïtes, la forme infectieuse injecte par le moustique, et la phase hépatique, et le stade érythrocytaire qui a lieu dans le sang. L immunité naturelle ou induite après immunisation contre le stade pré-érythrocytaire est très complexe et est mediée par les anticorps, les cellules T, et les cytokines que ces derniers sécrètent après reconnaissance des antigènes parasitaires. Diverses études ont mis en évidence un effet inhibiteur des cytokines, comme l interféron (IFN)-g, Interleukine (IL)-1 et IL-6 sur le développement hépatique de différentes espèces de Plasmodium, in vitro et in vivo. Cependant, selon le système cellules hôtes/espèce de parasite étudiées, des effets contradictoires ont été observés, en particulier pour le TNF-alpha. En effet, des études précédentes ont montré que le TNF- alpha humain inhibe le développement intra-hépatique de Plasmodium berghei dans des hépatomes humains. Par opposition, le TNF-alpha murin lui n a pas d effet sur le développement du parasite de même parasites dans des hépatocytes de souris. Ceci nous a conduit à postuler que différents paramètres (cellules hôtes, origine de la cytokine, espèces parasitaires) peuvent conduire à des résultats complètement opposés ne permettant pas de définir le rôle exact de ces cytokines contre la phase hépatique de Plasmodium. Nous avons donc, lors de ce travail de thèse, essayé de clarifier le rôle de TNF-alpha sur la phase hépatique de Plasmodium, en fonction des différents paramètres décrits plus haut. Ce travail nous a permis de tester la relevance des différents modèles in vitro et de démontrer que le TNF-alpha est un puissant inhibiteur du développement hépatique de P. falciparum dans les primo-culture d hepatocytes humains, contrairement à ce qui est observe pour les parasites de rongeurs cultives dans des cellules de rongeurs. Nous avons de même étudié les effets inhibiteurs d une cytokine de la famille que le TNF-alpha, le TNF-beta ou lymphotoxine. Nous avons pour la première fois décrit une rôle inhibiteur pour cette cytokine dans le développement intra-hépatique de Plasmodium. Le deuxième sujet développé durant cette thèse, a porté sur la physiologie du sporozoïte. Il est connu que l infectivité des sporozoites disséqués de moustiques infectés est abolie après 30 mn d incubation in vitro à 37 C. Cependant in vivo, après piqûre par le moustique un pourcentage non négligeable de sporozoïtes restent dans la peau et peuvent même après 24 heures infectés le foie. Ceci suggèrent qu il existent in vivo des signaux permettant la survie du sporozoïte. Nous avons donc mis au point de nouvelles techniques d études utilisant des parasite exprimant la protéine GFP pour pouvoir quantifier la viabilité des sporozoïtes en terme d intégrité membranaire, de métabolisme, de capacité à infecter des souris après différentes conditions en présence ou en absence de cellules. Ceci nous a permis de mettre en évidence que l infectivité des sporozoïtes est un processus complexe dépendant de l interaction du parasite avec son milieu extérieur et les cellules avec lesquelles il interagit.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Minimal Role for the Circumsporozoite Protein in the Induction of Sterile Immunity by Vaccination with Live Rodent Malaria Sporozoites▿ †

Immunization with live Plasmodium sporozoites under chloroquine prophylaxis (Spz plus CQ) induces sterile immunity against sporozoite challenge in rodents and, more importantly, in humans. Full protection is obtained with substantially fewer parasites than with the classic immunization with radiation-attenuated sporozoites. The sterile protection observed comprised a massive reduction in the hepatic parasite load and an additional effect at the blood stage level. Differences in the immune responses induced by the two protocols occur but are as yet little characterized. We have previously demonstrated that in mice immunized with irradiated sporozoites, immune responses against the circumsporozoite protein (CSP), the major component of the sporozoite's surface and the leading malaria vaccine candidate, were not essential for sterile protection. Here, we have employed transgenic Plasmodium berghei parasites in which the endogenous CSP was replaced by that of Plasmodium yoelii, another rodent malaria species, to assess the role of CSP in the sterile protection induced by the Spz-plus-CQ protocol. The data demonstrated that this role was minor because sterile immunity was obtained irrespective of the origin of CSP expressed by the parasites in this model of protection. The immunity was obtained through a single transient exposure of the host to the immunizing parasites (preerythrocytic and erythrocytic), a dose much smaller than that required for immunization with radiation-attenuated sporozoites

Comparison of the amino acid sequences of the <i>P. berghei</i> ANKA clone cy17 (34) and <i>P. falciparum</i> Welcome strain (35) CSP polypeptides.

<p>Black dots represent identical amino acid residues while bars represent amino acid residues with similar characteristics. The repeat regions (including the pre- and post-repeat sequences) are underlined.</p

Sterile protection in mice immunized with <i>P. berghei</i> irradiated sporozoites and challenged with <i>P. berghei</i> or <i>P. berghei</i> [<i>PfCS</i>] sporozoites.

<p>Mice were immunized with 1 or 3 injections of <i>P. berghei</i> (indicated on the left of the panel) before challenge with 5 000 <i>P. berghei</i> or <i>P. berghei</i> [<i>PfCS</i>] sporozoites. All naive control mice developed a patent blood-stage infection. The data are representative of those obtained in duplicate experiments.</p

Sterile protection in [BALB/c×C57BL/6] F1 mice immunized three times with <i>P. berghei</i> or <i>P. berghei</i> [<i>PfCS</i>] and challenged with 5000 <i>P. berghei</i> or <i>P. berghei</i> [<i>PfCS</i>] sporozoites.

<p>All animal groups (5 mice per group) were monitored for blood-stage infections by examination of Giemsa-stained blood smears obtained daily from day 2 to day 11 post-challenge. All naive control mice developed a patent blood-stage infection.</p