Development of nebulized inhalation delivery for fusion-inhibitory lipopeptides to protect non-human primates against Nipah-Bangladesh infection

Abstract

Nipah virus (NiV) is a lethal zoonotic paramyxovirus that can be transmitted from person to person through the respiratory route. There are currently no licensed vaccines or therapeutics. A lipopeptide-based fusion inhibitor was developed and previously evaluated for efficacy against the NiV-Malaysia strain. Intraperitoneal administration in hamsters showed superb prophylactic activity and promising efficacy, however the intratracheal delivery mode in non-human primates proved intractable and spurred the development of an aerosolized delivery route that could be clinically applicable. We developed an aerosol delivery system in an artificial respiratory 3D model and optimized the combinations of flow rate and particle size for lung deposition. We characterized the nebulizer device and assessed the safety of lipopeptide nebulization in an African green monkey model that mimics human NiV infection. Three nebulized doses of fusion-inhibitory lipopeptide were administered every 24 h, resulting in peptide deposition across multiple regions of both lungs without causing toxicity or adverse hematological and biochemical effects. In peptide-treated monkeys challenged with a lethal dose of NiV-Bangladesh, animals retained robust levels of T and B-lymphocytes in the blood, infection-induced lethality was significantly delayed, and 2 out of 5 monkeys were protected from NiV infection. The present study establishes the safety and feasibility of the nebulizer delivery method for AGM studies. Future studies will compare delivery methods using next-generation fusion-inhibitory anti-NiV lipopeptides to evaluate the potential role of this aerosol delivery approach in achieving a rapid antiviral response.

Keywords: Antivirals; Inhibitory lipopeptides; Nebulization; Nipah virus; Non-human primates; Virus-cell-fusion.

Fig. 1.

Experimental set up for the measurement of aerosol deposition using a 3D print cast AGM model. (A) Computed tomography (CT-Scan) of AGM upper airway sagittal view, (B) (C) Numerical 3D cast model from CT-scan; (D) Experimental set up of 3D print AGM cast, with respiratory pump, 3D cast and nebulizer with face mask. (E) Prediction of lung deposition using a breathing demonstrator, considering different breathing modes and aerosol particle sizes. (F) Influence of flow rate and particles sizes on lung deposition analyzed using the breathing demonstrator.

Fig. 2.

VIKI-PEG4-Chol lipopeptides remain effective after nebulization. (A) Schematic representation of the VIKI lipopeptide; (B) Lipopeptide was nebulized and harvested with an impactor. Serial 3-fold dilutions were performed and analyzed in a fusion assay based on a β-gal complementation assay. (C) Infection inhibition assay based on evaluation of plaque reduction, using 50 PFU of NiV. (D) Cytotoxicity assay was performed by assessing cell viability after 96h in an MTT assay.

Fig. 3.

Deposition of lipopeptides in lungs of AGMs following administration via nebulizer with no measurable toxic effect. (A) Schematic representation of the in vivo biodistribution/toxicology study. (B–C) Peptide deposition in the lungs of AGMs treated with nebulized peptide under anesthesia 24h before euthanasia. Staining was performed with rabbit anti-VIKI peptide and goat anti-rabbit Alexa 555, and DAPI was used to stain nuclei. Three AGMs were treated with nebulized VIKI peptide and one with saline solution. Fluorescence intensity of DAPI and VIKI stainings were evaluated using ImageJ software. (D–E) Body weight and body temperature after single or triple treatment with 4 mg of nebulized lipopeptide or with saline.

Fig. 4.

Effect of treatment with nebulized VIKI lipopeptide on NiV-infected AGMs. (A) Experimental design: animals were either treated wtih nebulized mock preparation or with 3 mL of VIKI lipopeptide (4 mg/kg), 24h and 6h before and 24h after intratracheal infection with 100 PFU of NiV. Blood samples were taken on indicateded days (red lines) and animals were followed daily. (B) Kaplan-Meier survival curves of the challenge experiment [VIKI-treated n = 5, and control (CTRL) composed of saline-nebulized animals (n = 3) and untreated historical cohort challenged with 100 PFU, (n = 5)]. Statistical difference analyzed by log-rank Mantel-Cox test. (C) NiV-N RNA quantification by RT-qPCR in animal lung, brain and spleen, obtained after autopsy of control group [n = 2 + 2 historical controls (Pastor et al., 2024)] and treated group (n = 5). (D) Quantification of viral RNA in pharyngeal swabs and Peripheral Blood Leukocytes (PBLs) from AGMs at different time points after infection. (E) Images of representative AGM lung sections from animals surviving or not surviving NiV infection, in haematoxylin-eosin saffron staining (HES, scale bars: 500 μm) and by immunofluorescence using a rabbit anti-NiV N antibody (scale bars: 50 μm). NiV N is marked in red, and nuclei are stained in blue with DAPI. Red blood cells display a green autofluorescence, highlighting the lung architecture. (F) Cytofluorometric analysis of the blood from infected AGMs. CD3⁺ T-lymphocytes, CD20⁺ B-lymphocytes and CD14⁺ monocytes levels were evaluated in blood at time points indicated in 4A and at the time of euthanasia. Results from CTRL group (n = 5) were compared to those from the VIKI group (n = 5). Curves represent the mean of each cell population for each group and the area within filled error bands the standard deviation. Statistical significance was analyzed at 7 dpi, corresponding to the time when all the animals were still alive, and results tested by a t-test, followed by a Mann-Whitney test; (*p = 0.05, **p < 0.01).