Malaria transmission-blocking vaccine candidate antigens have been developed to induce antibodies using different delivery systems, mainly protein-in-adjuvant formulations, independently in various laboratories giving varied transmission-blocking activity (TBA). However, only one candidate antigen has been tested in clinical trials. In order to advance the most efficacious target(s) for possible clinical development, a rank order of the leading antigens based on TBA in a head-to-head comparison using a single delivery platform was made. Candidate antigens, AnAPN1, PfsHAP2, Pfs230-C, Pfs25, and Pfs48/45 (with or without N-glycosylation site substitution), were generated as recombinant viral-vectored vaccines using simian adenovirus and modified vaccinia Ankara and administered to mice in a heterologous prime-boost regimen. Vaccine-induced antibody responses were induced to all except PfsHAP2 were maintained up to ten and a half months post-boost. TBA was assessed at the peak response against Plasmodium falciparum NF54 laboratory strain and African field isolates by ex vivo membrane feeding assays in Anopheles stephensi and A. gambiae respectively. Antibodies to three antigens [Pfs230-C, Pfs25 and Pfs48/45+_NGln] had TBA against P. falciparum NF54, and those against Pfs230-C and Pfs25 consistently showed efficacy regardless of the parasite exposure in both mosquito species. Further analysis of antibody responses to these two candidate antigens showed concentration-dependent efficacy against P. falciparum field isolates. In a rabbit study, responses to Pfs230-C, Pfs25 and Pfs48/45+_NGln also showed IgG concentration-dependent efficacy. To assess TBA against AnAPN1, antibody responses to three fragments were tested. TBA was observed only against N-terminal 135 amino acid fragment. Pfs230-C and Pfs25 were generated as fusion vaccines using either a self-cleaving or glycine-proline linker sequence. Comparable antibody responses were induced between the two fusion strategies that had synergistic effects at inhibiting P. falciparum NF54 development in A. stephensi.

Malária é um dos principais problemas de saúde nos países em desenvolvimento sendo o Plasmodium o agente etiológico da doença. Neste trabalho foi investigada a função do Ca2+ e da sinalização purinérgica na modulação proteolítica de Plasmodium. Utilizando peptídeos com apagamento intracelular de fluorescência (FRET) analisamos a atividade proteolítica ativada por Ca2+ liberado do retículo endoplasmático ou de compartimentos ácidos e investigamos as diferentes classes de proteases envolvidas. Utilizando-se P. berghei e P. yoelii verificou-se a importância do Ca2+ na modulação proteolítica além de diferenças fisiológicas nesta modulação dentre as espécies estudadas. Foram também investigados os efeitos de ATP, adenosina e GTP extracelular na proteólise e conclui-se que receptores purinérgicos estão envolvidos na habilidade do parasita ativar proteólise intracelular. Na terceira parte da tese foi estabelecido um modelo murino nocaute para analisar a relação Plasmodium-hospedeiro e propõem-se a interação do receptor InsP3R2 e a proteína PbRACK do parasita. / Malaria is a major health problem in developing countries Here we investigate the role of Ca2+ and purines in Plasmodium protease modulation. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca2+from endoplasmatic reticulum or acidic compartments and investigated the classes of affected proteases. Experiments in P. berghei and P. yoelii indicated a fundamental role for calcium in modulating proteolysis and points out key differences in proteolytic responses between Plasmodium species. We also investigated the effects of extracellular ATP, adenosine and GTP on triggering proteolysis. The data lead us to conclude that purinergic receptor is involved in the ability of the parasite to activate intracellular proteolysis by sensing external molecules. The third part of the thesis established a new murine knockout model to analyze Plasmodium-host signaling and suggest a possible interaction between InsP3R2 receptor and the PbRACK parasite protein.

Malária é um dos principais problemas de saúde nos países em desenvolvimento sendo o Plasmodium o agente etiológico da doença. Neste trabalho foi investigada a função do Ca2+ e da sinalização purinérgica na modulação proteolítica de Plasmodium. Utilizando peptídeos com apagamento intracelular de fluorescência (FRET) analisamos a atividade proteolítica ativada por Ca2+ liberado do retículo endoplasmático ou de compartimentos ácidos e investigamos as diferentes classes de proteases envolvidas. Utilizando-se P. berghei e P. yoelii verificou-se a importância do Ca2+ na modulação proteolítica além de diferenças fisiológicas nesta modulação dentre as espécies estudadas. Foram também investigados os efeitos de ATP, adenosina e GTP extracelular na proteólise e conclui-se que receptores purinérgicos estão envolvidos na habilidade do parasita ativar proteólise intracelular. Na terceira parte da tese foi estabelecido um modelo murino nocaute para analisar a relação Plasmodium-hospedeiro e propõem-se a interação do receptor InsP3R2 e a proteína PbRACK do parasita. / Malaria is a major health problem in developing countries Here we investigate the role of Ca2+ and purines in Plasmodium protease modulation. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca2+from endoplasmatic reticulum or acidic compartments and investigated the classes of affected proteases. Experiments in P. berghei and P. yoelii indicated a fundamental role for calcium in modulating proteolysis and points out key differences in proteolytic responses between Plasmodium species. We also investigated the effects of extracellular ATP, adenosine and GTP on triggering proteolysis. The data lead us to conclude that purinergic receptor is involved in the ability of the parasite to activate intracellular proteolysis by sensing external molecules. The third part of the thesis established a new murine knockout model to analyze Plasmodium-host signaling and suggest a possible interaction between InsP3R2 receptor and the PbRACK parasite protein.

Dendritic cells (DC) play a key role in immunity and represent a great target for modulation, because of their ability to prime T cells and direct their polarisation into effector subsets. Ticks release immunomodulatory compounds in their saliva, possibly in order to evade host immune responses during feeding. We have recently reported that Rhipicephalus appendiculatus ticks produce ‘Japanin’, a secretory lipocalin that arrests differentiation of monocytes into DC and reprogrammes maturation of DC in response to various stimuli towards a tolerogenic phenotype . Japanin was cloned and recombinantly expressed in a baculovirus system for subsequent immunological and biochemical analysis. This study was set out to further investigate the immunomodulatory activity of Japanin as well as the underlying mechanism of action. We have discovered that Japanin prevents DC-mediated proliferation and polarisation of allogeneic T cells. Experiments with labelled Japanin have demonstrated that it binds predominantly to ex vivo generated human monocyte-derived DC (moDC) and to a reduced degree to monocyte and DC populations in peripheral blood, yet to no other blood leucocytes. We have identified CD206, also known as the mannose receptor, as a Japanin-binding receptor on moDC. This identification has been achieved by crosslinking and subsequent pull-down of Japanin-receptor complexes from moDC. Affinity studies with recombinant CD206 constructs have confirmed the binding to Japanin. Moreover, the binding has been verified by specific siRNA knock-down of CD206 in moDC, which resulted in significantly decreased binding of Japanin. Unexpectedly, CD206 has appeared to be dispensable for at least most of the DC-modulatory activity of Japanin. Therefore, attempts were made to determine other factors in the mode of action of Japanin, through which we have found that IL-10 is not essentially involved. Further results have suggested that the activity of Japanin demands cell contact. Collectively, we have come to the conclusion that the mechanism of action of Japanin might require internalisation by DC, potentially enabling modulation of intracellular pathways involved in the regulation of DC maturation.