Fig. 1: Intranasal vaccination of Syrian hamsters with a maximum dose of OTS-228 confirms attenuation and immunogenicity.

a Experimental setup: Syrian hamsters (n = 14) were intranasally vaccinated with the maximum applicable dose of 10^6.1 TCID₅₀ of OTS-228 per animal. At 1 dpv, serologically naïve direct contact animals (n = 3) were co-housed with vaccinated animals in a 1:3 ratio to detect transmission events. b Survival rate and (c) body weight were monitored over 14 dpv, confirming no mortality and no weight loss. d Nasal wash samples were analyzed via RdRp (nsp12)-specific qPCR to determine genome copies per mL (gc/mL). Genome copy numbers were calculated based on a standard of known concentration. No viral genome shedding was detected in contact animals throughout the experiment. Bars indicate mean with SD. e Tissue sampling: Five vaccinated hamsters were euthanized at 5 dpv, and (f) the remaining nine vaccinated and three contact hamsters were euthanized at 14 dpv to assess genome copies in respiratory tract organ samples. At 5 dpv, virus genome was detected in nasal conchae and lung samples, but by 14 dpv, viral genome was completely cleared from lung samples and absent in contact animals. Mean is indicated by line. g Histopathology at 5 dpv showed no pneumonia-related atelectasis, shown as percentage of affected area. Representative hematoxylin-eosin stained sections showed the lack of atelectasis (whole slide image, scale bar 2.5 mm), along with interstitial macrophage infiltrates (green asterisk), perivascular (green arrow), and peribronchial immune cell infiltration (green arrowhead) (detailed images, scale bar 100 µm). h Antigen score and immunohistochemistry: Representative anti-SARS-CoV N-protein immunohistochemistry of lung sections showed multifocal virus antigen presence in type I pneumocytes (green arrowhead) and bronchial epithelial cells (green arrow) associated with the peribronchial and interstitial immune cell infiltration, shown in a consecutive slide of Fig. 1g, right lower image (scale bar 100 µm). i Serum antibodies specific to the SARS-CoV-2 RBD domain were detected in all vaccinated animals at 14 dpv, while absent in contact animals. j Virus neutralization test (VNT₁₀₀): The sera from vaccinated animals also exhibited neutralizing capacity in a virus neutralization test.