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

TL;DR

A single dose of an antibody targeting a specific protein on a parasite can cure a livestock disease in mice, offering a new treatment approach.

Contribution

Demonstration that an unmodified antibody can cure T. vivax infection in a murine model with a single dose.

Findings

A single dose of an anti-IFX monoclonal antibody cures T. vivax infection in mice.

No parasite reservoirs remain detectable in peripheral tissues after treatment.

Structural modeling identifies targetable regions on IFX for improved vaccine design.

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.

Trypanosoma 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.

Figures

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.

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).

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).

Full text

INTRODUCTION

The livelihoods of millions of people living in Africa are at risk due to infectious diseases that affect the health of their livestock, which provide them with essential food, milk, clothing, manure, and draught power. One major livestock disease is animal African trypanosomiasis (AAT), which is caused by blood-dwelling parasites of the genus Trypanosoma that affect many important farm animals, including sheep, pigs, and especially cattle (1). The impact of this disease is significant: over 3 million cattle die from this disease annually, with a direct economic cost to the African economy amounting to many hundreds of millions of dollars (2). This disease, therefore, represents a major constraint on the economic development of the mainly agrarian societies in African countries south of the Sahara (3, 4).

Although several species of trypanosome parasite can cause this disease in animals, the two most prevalent are Trypanosoma congolense and Trypanosoma vivax. The disease is currently managed by controlling the tsetse fly vector population and the use of trypanocidal drugs (5). The most commonly used drugs for therapy and prophylaxis are diminazene aceturate and isometamidium chloride: both were developed over 70 years ago in the 1950s (6, 7). These drugs can cause toxic side effects, and when inappropriately administered, for example, by underdosing, they have led to the emergence of drug resistance, which has now been reported in 21 out of the 37 countries in which the disease is endemic (8). The development of new trypanocides for livestock has lacked sufficient investment because there is a poor economic case: new drugs remain expensive to develop, and a price point that would provide a satisfactory return for developers could not be met by the primary customers, who are largely subsistence farmers. Despite this, there has been some encouraging recent progress achieved by the repurposing of drugs developed to treat human African trypanosomiasis (9–11).

Another control tool would be the development of an effective vaccine, but none yet exists for AAT (12). Vaccination has generally been considered a low-value option for this disease because the parasite has evolved sophisticated immunoregulatory mechanisms, such as antigenic variation (13), the ability to rapidly clear surface-bound host antibodies (14), and disruption of B-cell development and maintenance (15, 16), all of which enable trypanosomes to thrive in the blood of their infected host. We have previously shown that it is possible to induce protective immunity in an experimental model of T. vivax infection by eliciting an antibody response to an invariant flagellum-associated antigen called IFX prior to challenge by the parasite (17). In our experiments to determine the immunological mechanisms of protection, we identified a small panel of monoclonal antibodies, some of which could confer protection against T. vivax infection by passive transfer. While the original hybridoma-derived IgG1-isotype antibodies conferred only modest protection, reformatting the isotype of one antibody called 8E12 as an immune-effector-recruiting IgG2a isotype could elicit very potent protection when delivered prophylactically. We additionally demonstrated that by mutating the C1q and FcR binding sites in the Fc region of the antibody, we showed that complement recruitment was the major protective mechanism, followed by FcR binding, and lastly direct opsonization of the IFX antigen (17).

The passive transfer of immune serum from convalescent patients to treat and prevent infectious diseases has a long history of success (18). This approach, however, poses challenges in the clinic because it requires a sufficiently large number of donors, often varies in its efficacy, and carries the risk of unintentionally transferring infections. Success in treating a range of diseases from cancer to autoimmune disease using monoclonal antibody therapeutics has driven biotechnological progress that has made the isolation and large-scale production of antibody-based drugs rapid and cost-effective (19). Increasingly, this class of drugs has been developed for the treatment and prevention of infectious diseases (20). For example, several antibody-based drugs targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein were developed within a few months during the COVID-19 pandemic, including bamlanivimab, sotrovimab, and regdanvimab (21, 22). A major advantage of these drugs is their long pharmacokinetics, lack of toxicity, and generalized production methods (23). As well as treating viral infections, this strategy has also been shown to work as both therapy and prophylaxis for bacterial infections (22, 24). Importantly, antibody-based therapies have also been used to treat and prevent parasitic infections. This includes antibodies that target the circumsporozoite protein of sporozoite-stage Plasmodium falciparum parasites, which showed dose-dependent prevention of infection in Malian patients over a period of 6 months (25, 26). Similarly, experimental Trypanosoma brucei infections could be cured with a single dose of an antibody-drug conjugate targeting the haptoglobin-hemoglobin receptor (27).

Here, we investigate the possibility of using a monoclonal antibody targeting the T. vivax vaccine antigen IFX in a drug-like manner. We use structural modeling to identify the epitope of the 8E12 antibody and show, using a murine infection model, that a single dose of the unmodified antibody can cure an established T. vivax infection.

RESULTS

Structural modeling identified the location of the 8E12 epitope on IFX

To explore the possibility of using the IFX protein as a potential drug target, we first characterized the anti-IFX 8E12 monoclonal antibody that could elicit passive protection against T. vivax infections in more detail. We sequenced the rearranged light and heavy chains of the 8E12 antibody, which had been amplified from a hybridoma to create a single plasmid (28). Sequencing of this plasmid revealed kappa light chain usage and identified the amino acids forming the antibody paratope (Fig. S1). In our original study, we determined the 8E12 binding affinity as only moderate (KD = 14 nM), and mapped the 8E12 epitope to the N-terminal 168 amino acids of IFX by expressing fragments of the IFX ectodomain (17). To characterize the 8E12 epitope further, we first asked if the epitope was conformational by denaturing the IFX protein by heat treatment and reduction. We observed that while some of the binding activity was lost upon both heat treatment and protein reduction, not all binding activity was lost, indicating that the 8E12 epitope was at least partially dependent on protein folding (Fig. 1a). There is no experimental structural information on IFX, but recent advances in protein structure (29) and binding interface prediction (30) presented the opportunity to model both the IFX structure and predict the 8E12 epitope. Structural modeling suggests that the extracellular region of IFX adopts a dumbbell shape composed of two groups of alpha-helices joined by a long central helix (Fig. 1b). By using the sequence of IFX and the light and heavy chains of the 8E12 antibody, we used Alphafold3 to predict the location of possible epitopes. Of the five most highly-ranked models, all converged on a region of IFX at the tip of one of the helical clusters (Fig. 1b). We were reassured by the convergence of these predictions with the experimentally known N-terminal location of the epitope, and the lack of overt steric clashes with the location of N-linked glycans.

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.

To experimentally validate these predictions, we identified two solvent-exposed glutamic acid residues (E154 and E157 of TvY486_0807240), which contributed hydrogen bonds in the 8E12 co-complex structure prediction, and mutated these to lysine. We showed that E154 and E157 contributed to the 8E12 epitope by showing that the mutant IFX protein was unable to bind the 8E12 antibody (Fig. 1c). Importantly, the mutant IFX retained the ability to bind other anti-IFX monoclonal antibodies, demonstrating the mutations did not cause significant misfolding of the IFX ectodomain (Fig. 1c). Together, these data identify the 8E12 epitope to a membrane-distal tip of a helical cluster on the IFX ectodomain.

Repeated doses of 8E12 mAb can cure an experimental T. vivax infection

In our initial experiments, 8E12 was used prophylactically to prevent infection by administering the 8E12 antibody the day before, on the day, and the day after infection with T. vivax (17). To be used in a drug-like manner, the antibody must be able to clear an established infection. First, a bulk preparation of the 8E12 monoclonal antibody was produced by transfecting HEK293 cells and purified using immobilized protein G. The antibody was shown to be highly active by quantifying binding to recombinant IFX by enzyme-linked immunosorbent assay (ELISA) and over 95% pure by resolving the preparation on a protein gel (Fig. S2). Next, we infected mice with a transgenic form of T. vivax constitutively expressing the firefly luciferase and waited until 4 days post-infection, where a patent infection was apparent and could be quantified using bioluminescent imaging. At day 5 post-infection, the average bioluminescence was 4.1 × 10^7^ photon s^−1^, which corresponds to a parasitemia of ~1 × 10^6^ parasites per milliliter of blood, a value that is toward the highest level detected in infected cattle (31). From day 5, we administered a total of three daily doses of 50, 100, 200, or 400 µg of purified 8E12 IgG2a-formatted antibody and continued to measure parasitemia each day (Fig. 2a). We observed that the lowest (50 µg) doses, while slowing the multiplication of the parasite, were not able to clear the infection and all five animals were removed from the study by day 9 (Fig. 2b and c). While 100 µg doses could reduce parasitemia in most animals to very low levels by day 8, there was a recrudescence of the infection in each animal such that all had to be removed from the study by day 15 (Fig. 2b and c). At 200 µg, parasites were reduced below the limits of detection by day 7 (Fig. 2b), but then showed recrudescence of this infection that was highly variable between individuals. Interestingly, two of the five treated animals showed the ability to control the infection after day 20, which significantly exceeds the measured in vivo half-life for an IgG2a-isotype antibody, which has been measured at 6 to 8 days (32), suggesting that the animals had acquired some immunity. All animals receiving 400 µg doses showed no evidence of recrudescence with no detectable parasites from day 7 until beyond day 60.

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).

It is known that trypanosomes can reside in peripheral tissues, such as the brain and adipose tissue, which may have limited accessibility to antibody-based drugs. To ensure that there were no residual parasites that were being suppressed by the acquisition of immunity during the course of the experiment, we immunosuppressed apparently cured animals with two doses of cyclophosphamide at days 61 and 64 and observed the animals for a further 15 days. In no case did we observe any recrudescent infections. To further ensure that no parasite reservoirs remained, we dissected out the tissues of cured and immunosuppressed mice and subjected them to sensitive in vivo imaging. Again, we observed no parasite reservoirs in cured animals (Fig. 2d).

Cure of an experimental T. vivax infection with a single dose of 8E12 antibody

Cattle are the main target species for drugs that treat animal African trypanosomiasis, and because they usually are left to roam freely around the bush, and handling facilities are often rudimentary, being able to treat the disease with a single dose would be a major advantage. To determine whether animals could be cured by a single dose, we again infected groups of mice with T. vivax before administering a single dose of either 300, 400, or 600 µg of the 8E12-IgG2a-formatted antibody (Fig. 3a). To determine how rapidly parasites were killed, we imaged all animals at 4 and 24 h after the time of treatment. Consistent with our earlier finding that complement plays the major role in the protective immunological mechanism (17), we observed a dramatic reduction in parasitemia at 4 h, which progressed to below the limit of detection by 24 h (Fig. 3a and b). There was a recrudescence of the infection in all animals receiving the 300 µg dose, such that all animals were removed from the study by day 14. Two out of the five animals receiving the 400 µg dose were apparently cured of the infection, and a further two animals showed a drastic reduction in parasitemia by day 14, suggesting acquisition of adaptive immunity (Fig. 3a); two animals receiving this dose showed recrudescence after day 14 and were subsequently removed from the study. All animals receiving a single 600 µg dose were apparently sterilely cured. To demonstrate that there were no remaining parasites below the limits of detection, we immunosuppressed animals on day 48 and showed no recrudescence of the infection. Again, we dissected tissues of remaining immunosuppressed animals on day 57 and showed no evidence of residual parasites in peripheral tissues (Fig. 3c).

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).

DISCUSSION

Here, we have demonstrated that a single dose of a purified but otherwise unmodified monoclonal antibody can cure an established parasitic infection. This leads to the wider question of whether this general strategy of using antibody-based drugs could be a realistic treatment option for parasitic diseases of livestock. It is well known that the passive transfer of polyclonal immune serum from convalescent patients or immunized animals can be an effective treatment for infectious diseases and neutralizing toxins in humans (33–35). This approach is unlikely to be a practical option for an endemic disease of livestock because with an estimated 350 million heads of cattle in Africa, this number is impractically large (36). The general view is that therapeutic monoclonal antibodies are too expensive for use in livestock, but recent technological developments in isolating and producing large amounts of monoclonal antibodies driven by the multi-billion dollar therapeutic antibody industry have made these drugs much more affordable. The adoption of generalized production methods and economies of scale means that gram quantities of purified antibody can be produced for as little as $10 (37), and it is likely that these costs will continue to decrease. These factors have spurred the use of therapeutic antibodies to treat infectious diseases either by targeting susceptible pathogen proteins or even host proteins (38–40).

Clearly, there would be some challenges to the use of such a strategy for the treatment of AAT. Firstly, this disease is endemic in developing countries, where a reliable cold chain to ensure the protein-based product retains activity cannot be assumed. Advances in the design of heat-stable affinity scaffolds, however, provide confidence that this problem could be overcome (41, 42). Also, antibody-accessible cell surface proteins are often polymorphic so that specific antibody-based reagents will almost certainly be species-specific, meaning that a single drug would be unlikely to be effective against different species of trypanosome. IFX, for example, although highly conserved, is only found in T. vivax with no easily identifiable orthologues in other sequenced species (17). Because T. congolense and T. vivax are co-endemic in Africa, this would require the development of two different drugs, although a drug targeting just T. vivax would be useful in regions, such as South America, which has a large cattle industry and where T. vivax is the primary cause of AAT.

Administration of antibody doses that were not sufficient for cure typically resulted in a rapid reduction in parasitemia but that resulted in later recrudescence of the infection. In some animals, these later waves of parasitemia could be controlled, even more than 2 weeks after the last antibody dose. This suggests that the adaptive immune system had a role in controlling the infection at this stage because the half-life of circulating antibodies is typically around a week in mice. This may suggest that T. vivax infections do not significantly disable the adaptive immune system in the way that has been reported for T. brucei infections in mice (16, 43).

Targeting infectious diseases with antibody-based drugs does have some advantages. The antibody described here can be used in both a curative and prophylactic manner. This would be important for cattle farmers in Africa who generally lack adequate animal holding facilities, and so a single-dose drug that would cure current infections and provide lasting protection would be valuable and more practical to administer. Antibodies have excellent pharmacokinetics with circulating half-lives of several weeks due to their relatively large size and active recycling mechanisms mediated by the neonatal Fc receptor (FcRn) (44). Engineering of the antibody constant regions that interact with FcRn has enabled circulating half-lives to be significantly extended (45, 46). For use in cattle, it would be important to bovinize the antibody so that it effectively recruits immune effectors and does not elicit host anti-drug immune responses. The recent sequencing, annotation, and functional characterization of the bovine antibody locus now provide the necessary information to achieve this (47). Indeed, bovinization of the 8E12 antibody using a complement-recruiting cattle antibody isotype would be the next logical step to test to see if this strategy would be successful in cattle.

Motivated by the use of antibody-drug conjugates (ADCs) that have been used to treat cancer, research from MacGregor et al. showed that an experimental T. brucei infection could be cured with a single dose of an antibody targeting the haptoglobin-hemoglobin receptor conjugated to the toxin pyrrolobenzodiazepine (27). While initially conceived as a curative drug for human use, a similar ADC strategy could increase the potency of similar drugs for use in livestock. Here, though, there may be a conflict between trying to increase the in vivo half-life of the drug to provide prophylactic cover, and the need to ensure there is no long-lasting drug toxicity remaining in livestock products, such as the milk and meat, which would be undesirable for consumers. In summary, the work described here lends further support for the use of antibody-based drugs to treat livestock infectious diseases and identifies IFX as a new curative and preventative drug target for animal African trypanosomiasis.

MATERIALS AND METHODS

Mouse strains

Mice were maintained under a 12 h light/dark cycle at a temperature of 19°C–24°C and humidity between 40% and 65%. The mice used in this study were 6- to 14-week-old female Mus musculus strain BALB/c, which were obtained from a breeding colony at the Research Support Facility, Wellcome Sanger Institute.

Statistical analysis

The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment, although animal dosing and parasite quantification were performed by independent researchers.

Cell lines and antibodies

Recombinant proteins and antibodies used in this study were expressed in HEK293-6E cells provided by Y. Durocher. Cell lines were not authenticated but were tested for mycoplasma. The 8E12 antibody was cloned and expressed recombinantly as a mouse IgG2a isotype as described (17, 28). The mouse IgG2a isotype control antibody was C1.18.4, BioXcell Cat. No. BE0085. The other anti-IFX antibodies, 3D12, 6B3, 8C9, 10E2, and 2H3 were produced and purified by hybridoma supernatant as described (17). The antibody used for protein quantification in ELISAs was a mouse monoclonal anti-His (His-Tag monoclonal antibody, 70796, EMD-Millipore). The secondary antibody used was a goat anti-mouse alkaline phosphatase-conjugated secondary (A3562, Sigma-Aldrich).

Recombinant protein and antibody expression and purification

The recombinant soluble IFX ectodomain and controls were expressed in HEK293-6E cells as described (17). The IFX E154K/E157K mutant was produced using gene synthesis by Twist Biosciences. Proteins were expressed as enzymatically monobiotinylated proteins by co-transfection with a secreted version of the protein biotin ligase (BirA) as previously described (48). Supernatants were collected 5 days after transfection, filtered, and stored at 4°C until use. Proteins were purified using Ni^2+^ immobilized metal-ion affinity chromatography as described (49).

Enzyme-linked immunosorbent assays

To determine if the anti-IFX antibodies recognized a conformational epitope, the purified monobiotinylated ectodomain of IFX was denatured by heat treatment (90°C) in the presence of 10 millimoles of DTT for 10 min; once cooled, 20 millimoles of iodoacetamide was added. The immunoreactivity of the 8E12 monoclonal antibody to the entire ectodomain of wild-type IFX and the E154K/E157K mutant was quantified by ELISA. Purified ectodomains were normalized by dilution in PBS containing 0.2% Tween-20 (PBST) and 2% BSA and captured on a streptavidin-coated microtiter plate by incubating overnight at 4°C. Plates were washed two times with PBST, and once with PBS. Purified anti-IFX monoclonal antibodies were diluted in PBS/2% BSA, added to the ELISA plate, and incubated overnight at 4°C. A 1 in 1,000 dilution of an anti-6His antibody (EMD Millipore) was used as a control to ensure ectodomains were captured. The plate was washed two times with PBST, and once with PBS, before incubating for 1 h with a 1 in 5,000 dilution of anti-mouse IgG secondary antibody conjugated to alkaline phosphatase (Sigma-Aldrich). After two further washes with PBST, and one wash with PBS, 70 µL of 1 mg/mL Sigma 104 phosphatase substrate was added, and substrate hydrolysis was quantified at 405 nm using a plate reader (Spark, Tecan).

Structural modeling

The three amino acid sequences encoding the light and heavy chains of the 8E12 Fab region and the entire ectodomain of IFX (Uniprot: G0TZT9) were used as an input for AlphaFold3 for structural modeling. Models that exceeded the template modeling and interface predicted template modeling scores of 0.5 and 0.8, respectively, and with the highest ranking, predicted “Local Distance Difference Test” pLDDT scores were selected for residue mutation.

Quantification of T. vivax infections by bioluminescent in vivo imaging

Mice were infected with bloodstream forms of T. vivax parasites from the blood of an infected donor mouse at the peak of parasitemia. Parasites were diluted in PBS and 20 mM D-glucose, quantified by microscopy, and used to infect mice by intravenous injection. The luciferase substrate D-luciferin (potassium salt, Source BioScience) was resuspended to 30 mg mL^−1^ in Dulbecco’s PBS (Hyclone), filter-sterilized (0.22 µm), and stored in aliquots until use at −20°C. Luciferin was administered by intraperitoneal injection at 200 mg kg^−1^ 10 min prior to imaging. Mice moved freely for 3 min before being anesthetized (2.5% isoflurane) and placed in the imaging chamber, where anesthesia was maintained. The average background bioluminescence measurement was determined by luciferin administration in five uninfected female BALB/c mice and calculating the mean whole-body bioluminescence. This background value is indicated as a light gray shading on bioluminescence plots where appropriate. Long-term persistence of the parasites in different organs of infected mice was determined by imaging mice, which were then euthanized with an overdose of anesthetic. Mice were perfused with PBS until the perfusion fluid ran clear, the organs were dissected, and arranged on a Petri dish and bathed in PBS containing 20 mM glucose and 3.3 mg mL^−1^ luciferin. Emitted photons were acquired by a charge-coupled device camera (IVIS Spectrum Imaging System, Perkin Elmer). Regions of interest were drawn and total photons emitted from the image of each mouse were quantified using Living Image software version 4.7.4 (Xenogen); the results were expressed as the number of photons s^−1^. Bioluminescence values were exported and plotted in Prism GraphPad version 8.0.2, which was also used for testing statistical significance where needed. Persistence of potential parasite reservoirs in 8E12-IgG2a-treated mice was assessed by immunosuppression. Mice received two doses of cyclophosphamide (150 mg/kg, Cayman Chemical) 3 days apart using intraperitoneal injections and were monitored by imaging to assess parasite recrudescence.