Efficacy of ivermectin and its metabolites against Plasmodium falciparum liver stages in primary human hepatocytes.

Ivermectin, a broad-spectrum anti-parasitic drug, has been proposed as a novel vector control tool to reduce malaria transmission by mass drug administration. Ivermectin and some metabolites have mosquito-lethal effect, reducing Anopheles mosquito survival. Ivermectin inhibits liver stage development in a rodent malaria model, but no inhibition was observed in a primate malaria model or in a human malaria challenge trial. In the liver, cytochrome P450 3A4 and 3A5 enzymes metabolize ivermectin, which may impact drug efficacy. Thus, understanding ivermectin metabolism and assessing this impact on Plasmodium liver stage development is critical. Using primary human hepatocytes (PHHs), we characterized ivermectin metabolism and evaluated the efficacy of ivermectin and its primary metabolites M1 (3″-O-demethyl ivermectin) and M3 (4-hydroxymethyl ivermectin) against Plasmodium falciparum liver stages. Two different modes of ivermectin exposure were evaluated: prophylactic mode (days 0-3 post-infection) and curative mode (days 3-5 post-infection). We used two different PHH donors and modes to determine the inhibitory concentration (IC50) of ivermectin, M1, M3, and the known anti-malarial drug pyrimethamine, with IC50 values ranging from 1.391 to 14.44, 9.95-23.71, 4.767-8.384, and 0.9073-5.416 µM, respectively. In our PHH model, ivermectin and metabolites M1 and M3 demonstrated inhibitory activity against P. falciparum liver stages in curative treatment mode (days 3-5) and marginal activity in prophylactic treatment mode (days 0-3). Ivermectin had improved efficacy when co-administered with ketoconazole, a specific inhibitor of cytochrome P450 3A4 enzyme. Further studies should be performed to examine ivermectin liver stage efficacy when co-administered with CYP3A4 inhibitors and anti-malarial drugs to understand the pharmacokinetic and pharmacodynamic drug-drug interactions that enhance efficacy against human malaria parasites in vitro.

Comparative analyses of functional antibody-mediated inhibition with anti-circumsporozoite monoclonal antibodies against transgenic Plasmodium berghei.

BACKGROUND: Acquired functional inhibitory antibodies are one of several humoral immune mechanisms used to neutralize foreign pathogens. In vitro bioassays are useful tools for quantifying antibody-mediated inhibition and evaluating anti-parasite immune antibodies. However, a gap remains in understanding of how antibody-mediated inhibition in vitro translates to inhibition in vivo. In this study, two well-characterized transgenic Plasmodium berghei parasite lines, PbmCh-luc and Pb-PfCSP(r), and murine monoclonal antibodies (mAbs) specific to P. berghei and Plasmodium falciparum circumsporozoite protein (CSP), 3D11 and 2A10, respectively, were used to evaluate antibody-mediated inhibition of parasite development in both in vitro and in vivo functional assays. METHODS: IC50 values of mAbs were determined using an established inhibition of liver-stage development assay (ILSDA). For the in vivo inhibition assay, mice were passively immunized by transfer of the mAbs and subsequently challenged with 5.0 × 103 sporozoites via tail vein injection. The infection burden in both assays was quantified by luminescence and qRT-PCR of P. berghei 18S rRNA normalized to host GAPDH. RESULTS: The IC50 values quantified by relative luminescence of mAbs 3D11 and 2A10 were 0.396 µg/ml and 0.093 µg/ml, respectively, against transgenic lines in vitro. Using the highest (> 90%) inhibitory antibody concentrations in a passive transfer, an IC50 of 233.8 µg/ml and 181.5 µg/ml for mAbs 3D11 and 2A10, respectively, was observed in vivo. At 25 µg (250 µg/ml), the 2A10 antibody significantly inhibited liver burden in mice compared to control. Additionally, qRT-PCR of P. berghei 18S rRNA served as a secondary validation of liver burden quantification. CONCLUSIONS: Results from both experimental models, ILSDA and in vivo challenge, demonstrated that increased concentrations of the homologous anti-CSP repeat mAbs increased parasite inhibition. However, differences in antibody IC50 values between parasite lines did not allow a direct correlation between the inhibition of sporozoite invasion in vitro by ILSDA and the inhibition of mouse liver stage burden. Further studies are needed to establish the conditions for confident predictions for the in vitro ILSDA to be a predictor of in vivo outcomes using this model system.

Naturally acquired antibodies against Plasmodium vivax pre-erythrocytic stage vaccine antigens inhibit sporozoite invasion of human hepatocytes in vitro.

In Plasmodium vivax, the most studied vaccine antigens are aimed at blocking merozoite invasion of erythrocytes and disease development. Very few studies have evaluated pre-erythrocytic (PE) stage antigens. The P. vivax circumsporozoite protein (CSP), is considered the leading PE vaccine candidate, but immunity to CSP is short-lived and variant specific. Thus, there is a need to identify other potential candidates to partner with CSP in a multivalent vaccine to protect against infection and disease. We hypothesize that sporozoite antigens important for host cell infection are considered potential targets. In this study, we evaluated the magnitude and quality of naturally acquired antibody responses to four P. vivax PE antigens: sporozoite surface protein 3 (SSP3), sporozoite protein essential for traversal 1 (SPECT1), cell traversal protein of ookinetes and sporozoites (CelTOS) and CSP in plasma of P. vivax infected patients from Thailand. Naturally acquired antibodies to these antigens were prevalent in the study subjects, but with significant differences in magnitude of IgG antibody responses. About 80% of study participants had antibodies to all four antigens and only 2% did not have antibodies to any of the antigens. Most importantly, these antibodies inhibited sporozoite infection of hepatocytes in vitro. Significant variations in magnitude of antigen-specific inhibitory antibody responses were observed with individual samples. The highest inhibitory responses were observed with anti-CelTOS antibodies, followed by anti-SPECT1, SSP3 and CSP antibodies respectively. These data highlight the vaccine potential of these antigens in protecting against hepatocyte infection and the need for a multi-valent pre-erythrocytic vaccine to prevent liver stage development of P. vivax sporozoites.