Danny M. van Willigen

Despite promising results in malaria-naïve individuals, whole sporozoite (SPZ) vaccine efficacy in malaria-endemic settings has been suboptimal. Vaccine hypo-responsiveness due to previous malaria exposure has been posited as responsible, indicating the need for SPZ vaccines of increased immunogenicity. To this end, we here demonstrate a proof-of-concept for altering SPZ immunogenicity, where supramolecular chemistry enables chemical augmentation of the parasite surface with a TLR7 agonist-based adjuvant (SPZ-SAS(CL307)). In vitro, SPZ-SAS(CL307) remained well recognized by immune cells and induced a 35-fold increase in the production of pro-inflammatory IL-6 (p < 0.001). More promisingly, immunization of mice with SPZ-SAS(CL307) yielded improved SPZ-specific IFN-γ production in liver-derived NK cells (percentage IFN-γ⁺ cells 11.1 ± 1.8 vs. 9.4 ± 1.5%, p < 0.05), CD4⁺ T cells (4.7 ± 4.3 vs. 1.8 ± 0.7%, p < 0.05) and CD8⁺ T cells (3.6 ± 1.4 vs. 2.5 ± 0.9%, p < 0.05). These findings demonstrate the potential of using chemical augmentation strategies to enhance the immunogenicity of SPZ-based malaria vaccines.

Background: The occurrence of accidental nerve damage during surgery and the increasing application of image guidance during head-and-neck surgery have highlighted the need for molecular targeted nerve-sparing interventions. The implementation of such interventions relies on the availability of nerve-specific tracers. In this paper, we describe the development of a truncated peptide that has an optimized affinity for protein zero (P0), the most abundant protein in myelin. Methods and Materials: Further C- and N-terminal truncation was performed on the lead peptide Cy5-P0_101–125. The resulting nine Cy5-labelled peptides were characterized based on their photophysical properties, P0 affinity, and in vitro staining. These characterizations were combined with evaluation of the crystal structure of P0, which resulted in the selection of the optimized tracer Cy5-P0_112–125. A near-infrared Cy7-functionalized derivative (Cy7-P0_112–125) was used to perform an initial evaluation of fluorescence-guided surgery in a porcine model. Results: Methodological truncation of the 26-amino-acid lead compound Cy5-P0_101–125 resulted in a size reduction of 53.8% for the optimized peptide Cy5-P0_112–125. The peptide design and the 1.5-fold affinity gain obtained after truncation could be linked to interactions observed in the crystal structure of the extracellular portion of P0. The near-infrared analogue Cy7-P0_112–125 supported nerve illumination during fluorescence-guided surgery in the head-and-neck region in a porcine model. Conclusions: Methodological truncation yielded a second-generation P0-specific peptide. Initial surgical evaluation suggests that the peptide can support molecular targeted nerve imaging.

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and ¹¹¹In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [¹¹¹In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.

Type 2 immunity plays an essential role in the maintenance of metabolic homeostasis and its disruption during obesity promotes meta-inflammation and insulin resistance. Infection with the helminth parasite Schistosoma mansoni and treatment with its soluble egg antigens (SEA) can induce a type 2 immune response in metabolic organs and improve insulin sensitivity and glucose tolerance in obese mice, yet a causal relationship remains unproven. Here, we investigated the effects and underlying mechanisms of the T2 ribonuclease omega-1 (ω1), one of the major S. mansoni immunomodulatory glycoproteins, on metabolic homeostasis. Male C57Bl6/J mice were fed a high-fat diet for 12 weeks followed by bi-weekly injection of SEA, ω1 or vehicle for 4 additional weeks. Whole-body metabolic homeostasis and energy expenditure were assessed by glucose/insulin tolerance tests and indirect calorimetry, respectively. Tissue-specific immune cell phenotypes were determined by flow cytometry. We show that treatment of obese mice with plant-produced recombinant ω1, harboring similar glycan motifs as present on the native molecule, decreased body fat mass and improved systemic insulin sensitivity and glucose tolerance in a time-and dose-dependent manner. This effect was associated with an increase in white adipose tissue (WAT) type 2 T helper cells, eosinophils and alternatively-activated macrophages, without affecting type 2 innate lymphoid cells. In contrast to SEA, the metabolic effects of ω1 were still observed in obese STAT6-deficient mice with impaired type 2 immunity, indicating that its metabolic effects are independent of the type 2 immune response. Instead, we found that ω1 inhibited food intake, without affecting locomotor activity, WAT thermogenic capacity or whole-body energy expenditure, an effect also occurring in leptin receptor-deficient obese and hyperphagic db/db mice. Altogether, we demonstrate that while the helminth glycoprotein ω1 can induce type 2 immunity, it improves whole-body metabolic homeostasis in obese mice by inhibiting food intake via a STAT6-independent mechanism. Author summary: The obesity-induced chronic low-grade inflammation, notably in adipose tissue, contributes to insulin resistance and increased risk of type 2 diabetes. We have previously shown that infection with parasitic helminth worms was associated with protection against obesity-related metabolic dysfunctions both in mice and humans. We have also reported that treatment of obese mice with an extract of Schistosoma mansoni eggs (SEA) improves insulin sensitivity and glucose tolerance, a beneficial effect that was associated with a helminth-specific type 2 immune response in metabolic organs. Here, we studied the effects of omega-1 (ω1), a single immunomodulatory molecule from SEA, on metabolic health in obese mice, and investigated the role of the host immune response elicited. We found that ω1 induced a helminth-characteristic type 2 immune response in adipose tissue and improved both insulin sensitivity and glucose tolerance in obese mice. Yet, in contrast to SEA, ω1’s immunomodulatory properties were dispensable for its metabolic effects. Instead, we show that ω1 inhibited food intake, a feature accounting for most of the improvements in metabolic health. Together, our findings indicate that helminth molecules may improve metabolic health through multiple distinct mechanisms, and further characterization of such molecules could lead to new therapeutic strategies to combat obesity.

The availability of a receptor for theranostic pretargeting approaches was assessed by use of a new click‐chemistry‐based deactivatable fluorescence‐quenching concept. The efficacy was evaluated in a cell‐based model system featuring both membranous (available) and internalized (unavailable) receptor fractions of the clinically relevant receptor chemokine receptor 4 (CXCR4). Proof of concept was achieved with a deactivatable tracer consisting of a CXCR4‐specific peptide functionalized with a Cy5 dye bearing a chemoselective azide handle (N₃‐Cy5‐AcTZ14011). Treatment with a Cy7 quencher dye (Cy7‐DBCO) resulted in optically silent Cy7‐[click]‐Cy5‐AcTZ14011. In situ, a >90 % FRET‐based reduction of the signal intensity of N₃‐Cy5‐AcTZ14011 [K_D=(222.4±25.2) nm] was seen within minutes after quencher addition. In cells, discrimination between the membranous and the internalized receptor fraction could be achieved through quantitative assessment of quenching/internalization kinetics. Similar evaluation of an activatable tracer variant based on the same targeting moiety (Cy5‐S‐S‐Cy3‐AcTZ14011) was unsuccessful in vitro. As such, using the described deactivatable approach to screen membrane receptors and their applicability in receptor‐(pre‐)targeted theranostics can become straightforward.