Disclosure of cinnamic acid/4,9-diaminoacridine conjugates as multi-stage antiplasmodial hits.

4,9-diaminoacridines with reported antiplasmodial activity were coupled to different trans-cinnamic acids, delivering a new series of conjugates inspired by the covalent bitherapy concept. The new compounds were more potent than primaquine against hepatic stages of Plasmodium berghei, although this was accompanied by cytotoxic effects on Huh-7 hepatocytes. Relevantly, the conjugates displayed nanomolar activities against blood stage P. falciparum parasites, with no evidence of hemolytic effects below 100 µM. Moreover, the new compounds were at least 25-fold more potent than primaquine against P. falciparum gametocytes. Thus, the new antiplasmodial hits disclosed herein emerge as valuable templates for the development of multi-stage antiplasmodial drug candidates.

Variable long-term protection by radiation-, chemo-, and genetically-attenuated Plasmodium berghei sporozoite vaccines.

Long-term protection against malaria remains one of the greatest challenges of vaccination against this deadly parasitic disease. Whole-sporozoite (WSp) malaria vaccine formulations, which target the Plasmodium parasite's pre-erythrocytic stages, include radiation-attenuated sporozoites (RAS), early- and late-arresting genetically-attenuated parasites (EA-GAP and LA-GAP, respectively), and chemoprophylaxis with sporozoites (CPS). Although all these four vaccine formulations induce protective immune responses in the clinic, data on the longevity of the antimalarial protection they afford remain scarce. We employed a mouse model of malaria to assess protection conferred by immunization with P. berghei (Pb)-based surrogates of these four WSp formulations over a 36-week period. We show that EA-GAP WSp provide the lowest overall protection against an infectious Pb challenge, and that while immunization with RAS and LA-GAP WSp elicits the most durable protection, the protective efficacy of CPS WSp wanes rapidly over the 36-week period, most notably at higher immunization dosages. Analyses of liver immune cells show that CD44hi CD8+ T cells in CPS WSp-immunized mice express increased levels of the co-inhibitory PD-1 and LAG-3 markers compared to mice immunized with the other WSp formulations. This indicates that memory CD8+ T cells elicited by CPS WSp immunization display a more exhausted phenotype, which may explain the rapid waning of protection conferred by the former. These results emphasize the need for a detailed comparison of the duration of protection of different WSp formulations in humans and suggest a more beneficial effect of RAS and LA-GAP WSp compared to EA-GAP or CSP WSp.

The effect of dosage on the protective efficacy of whole-sporozoite formulations for immunization against malaria.

Immunization with Plasmodium sporozoites, either attenuated or administered under the cover of an antimalarial drug, can induce strong protection against malaria in pre-clinical murine models, as well as in human trials. Previous studies have suggested that whole-sporozoite (WSpz) formulations based on parasites with longer liver stage development induce higher protection, but a comparative analysis of four different WSpz formulations has not been reported. We employed a rodent model of malaria to analyze the effect of immunization dosage on the protective efficacy of WSpz formulations consisting of (i) early liver arresting genetically attenuated parasites (EA-GAP) or (ii) radiation-attenuated sporozoites (RAS), (iii) late arresting GAP (LA-GAP), and (iv) sporozoites administered under chemoprophylaxis, that are eliminated upon release into the bloodstream (CPS). Our results show that, unlike all other WSpz formulations, EA-GAP fails to confer complete protection against an infectious challenge at any immunization dosage employed, suggesting that a minimum threshold of liver development is required to elicit fully effective immune responses. Moreover, while immunization with RAS, LA-GAP and CPS WSpz yields comparable, dosage-dependent protection, protection by EA-GAP WSpz peaks at an intermediate dosage and markedly decreases thereafter. In-depth immunological analyses suggest that effector CD8+ T cells elicited by EA-GAP WSpz immunization have limited developmental plasticity, with a potential negative impact on the functional versatility of memory cells and, thus, on protective immunity. Our findings point towards dismissing EA-GAP from prioritization for WSpz malaria vaccination and enhance our understanding of the complexity of the protection elicited by these WSpz vaccine candidates, guiding their future optimization.

Impact of Dietary Protein Restriction on the Immunogenicity and Efficacy of Whole-Sporozoite Malaria Vaccination.

Malaria remains one of the world's most prevalent infectious diseases. Several vaccination strategies currently under investigation aim at hampering the development of the Plasmodium parasite during the clinically silent liver stage of its life cycle in the mammalian host, preventing the subsequent disease-associated blood stage of infection. Immunization with radiation-attenuated sporozoites (RAS), the liver-infecting parasite forms, can induce sterile protection against malaria. However, the efficacy of vaccine candidates in malaria-naïve individuals in high-income countries is frequently higher than that found in populations where malaria is endemic. Malnutrition has been associated with immune dysfunction and with a delay or impairment of the immune response to some vaccines. Since vaccine efficacy depends on the generation of competent immune responses, and malaria-endemic regions are often associated with malnutrition, we hypothesized that an inadequate host nutritional status, specifically resulting from a reduction in dietary protein, could impact on the establishment of an efficient anti-malarial immune response. We developed a model of RAS immunization under low protein diet to investigate the impact of a reduced host protein intake on the immunogenicity and protective efficacy of this vaccine. Our analysis of the circulating and tissue-associated immune compartments revealed that a reduction in dietary protein intake during immunization resulted in a decrease in the frequency of circulating CD4+ T cells and of hepatic NK cells. Nevertheless, the profile of CD8+ T cells in the blood, liver and spleen was robust and minimally affected by the dietary protein content during RAS immunization, as assessed by supervised and in-depth unsupervised X-shift clustering analysis. Although mice immunized under low protein diet presented higher parasite liver load upon challenge than those immunized under adequate protein intake, the two groups displayed similar levels of protection from disease. Overall, our data indicate that dietary protein reduction may have minimal impact on the immunogenicity and efficacy of RAS-based malaria vaccination. Importantly, this experimental model can be extended to assess the impact of other nutrient imbalances and immunization strategies, towards the refinement of future translational interventions that improve vaccine efficacy in malnourished individuals.