Cure of experimental Trypanosoma vivax infection with a single dose of an unmodified antibody-based drug targeting the invariant flagellum cell surface protein IFX

Abstract

Animal African trypanosomiasis (AAT) is an infectious wasting disease of economically important livestock caused by Trypanosoma spp. parasites. The disease is primarily caused by two species: Trypanosoma congolense and Trypanosoma vivax, which are endemic in many African countries. AAT is managed by therapeutic and prophylactic drugs; however, resistance is now widely reported, and the development of new drugs has been impeded due to a chronic lack of investment. Recently, we identified an invariant flagellar-associated cell surface protein (IFX) that could elicit protective immune responses when used as a vaccine against T. vivax. We showed that a complement-recruiting anti-IFX monoclonal antibody can prevent infection when used prophylactically. Here, we show that this same unmodified antibody can be used to cure T. vivax infections in a murine experimental model. Importantly, we show that infections can be treated with a single dose and demonstrate full cure by the lack of detectable parasites in peripheral tissues even after immunosuppression. Using structural modeling and site-directed mutagenesis, we localize the protective antibody epitope, thereby identifying targetable regions on IFX to improve vaccine design. Together, these findings validate IFX as both a prophylactic and curative drug target that could be useful in the management of AAT.IMPORTANCETrypanosoma vivax is a parasite that causes animal African trypanosomiasis (AAT), a chronic wasting disease that infects economically important livestock animals, which is a particular problem in African countries south of the Sahara. The impact of this disease is significant: it is responsible for over 3 million cattle deaths and an estimated $4.5 billion of annual lost productivity. There is a desperate need to develop new control measures because resistance is now widely reported to the drugs commonly used to treat this infection. We show here that a single dose of an unmodified monoclonal antibody that recognizes IFX-a parasite cell surface protein localized to the flagellum-is sufficient to cure an established T. vivax infection with no parasite reservoirs detectable in peripheral tissues. Our finding validates IFX as a new drug target and provides a rationale route to the development of new drugs to target AAT.

Keywords: Trypanosoma; drug development; host-parasite relationship; monoclonal antibodies; neutralizing antibodies.

Fig 1

Identifying the location of the anti-IFX 8E12 monoclonal antibody epitope by structural modeling and site-directed mutagenesis. (a) Biochemical characterization of anti-IFX monoclonal antibody epitopes. The monobiotinylated IFX ectodomain was immobilized in wells of a streptavidin-coated microtiter plate without treatment or after heat treatment with reduction. Immunoreactivity of antibodies 3D12, 6B3, and 8C9 was completely abrogated in the denatured protein, showing they recognized a conformational epitope; immunoreactivity to 2H3 and 10E2 was retained upon denaturation, demonstrating these antibodies recognized a non-conformational epitope. Immunoreactivity to 8E12 was only partly ablated by heat treatment and protein reduction, demonstrating that the epitope was only partly conformational. Bars represent means ± SD, n = 3. (b) Structural model of the IFX-8E12 Fab complex. The predicted structure of the extracellular region of IFX (green), including the location of modeled potential N-linked glycans (gray sticks) in complex with the 8E12 antibody Fab fragment, showing light chain (red) and heavy chain (blue). Inset: atomic details of the 8E12 epitope highlighting the position of glutamic acid residues at positions 154 and 157. The approximate location of the parasite plasma membrane phospholipids is shown in gray. (c) Identification of the epitope recognized by the 8E12 antibody. The entire extracellular region of IFX was expressed as either its wild-type form (left panel) or the E154K/E157K mutant (right panel) as a soluble biotinylated protein and immobilized in wells of a streptavidin-coated plate. Titrations of the indicated anti-IFX antibodies were tested for direct binding to the IFX proteins by enzyme-linked immunosorbent assay (ELISA). While both wild-type and mutant IFX bound antibodies 3D12, 6B3, and 8C9, the 8E12 antibody bound only the wild-type form.

Fig 2

Cure of an experimental T. vivax infection with the anti-IFX 8E12-IgG2a-formatted monoclonal antibody. (a) Schematic showing the experimental plan. Groups of five animals were infected with luciferase-expressing transgenic T. vivax parasites on day 0. Varying doses of the 8E12 monoclonal antibody were administered daily on days 5, 6, and 7, and parasitemia was quantified by bioluminescent imaging. Animals were immunosuppressed by two doses of cyclophosphamide (CPM). (b) Images of mice infected with bioluminescent T. vivax immediately before and 24 h after the last administration of the indicated doses of the 8E12 antibody. (c) Longitudinal analysis of individual infected animals with the indicated doses of 8E12 relative to an isotype-matched control antibody (Ctrl). Bioluminescence (solid lines) and survival (dashed lines) are plotted for each experimental group. Background level of bioluminescence is indicated by gray shading. Crosses indicate where animals were removed from the study for welfare reasons that are thought to be unrelated to the infection. Black and gray arrows represent doses of the 8E12 antibody and injection of the cyclophosphamide immunosuppressant, respectively. Hash symbols represent potential bioluminescence signal leakage from a heavily infected mouse to an adjacent mouse with a lower bioluminescence signal during image acquisition. (d) Lack of residual parasites in peripheral tissues of treated and immunosuppressed animals. No bioluminescent parasites were detected in the named tissues of a T. vivax-infected mouse that was administered with three 200 µg doses of the 8E12 antibody and subsequently treated with an immunosuppressant (left panels), relative to a control infected animal (right panels).

Fig 3

Rapid cure of an experimental T. vivax infection with a single dose of anti-IFX 8E12-IgG2a-formatted monoclonal antibody. (a) Longitudinal analysis of individual infected animals with the indicated single doses of 8E12 relative to an isotype-matched control antibody (Ctrl). Bioluminescence (solid lines) and survival (dashed lines) are plotted for each experimental group. Background level of bioluminescence is indicated by gray shading. Crosses indicate where animals were removed from the study for welfare reasons, thought to be unrelated to the infection. Black and gray arrows represent the single dose of 8E12 antibody and the injection of CPM immunosuppressant, respectively. Hash symbols represent potential bioluminescence signal leakage from a heavily infected mouse to an adjacent mouse with a lower bioluminescence signal during image acquisition. (b) Dorsal views of mice infected with bioluminescent T. vivax at 0 (days post-infection [Dpi] 5), 4 (Dpi 5–4 h), and 24 (Dpi 6) hours after the administration of the indicated doses of the 8E12 antibody showing rapid cure of infection. (c) Lack of residual parasites in peripheral tissues of treated and immunosuppressed animals. No bioluminescent parasites were detected in the named tissues of a T. vivax-infected mouse that was administered a single 400 µg dose of the 8E12 antibody and subsequently treated with an immunosuppressant (left panels), relative to a control infected animal (right panels).