Fig. 4: Intranasal vaccination of Syrian hamsters with low-dose OTS-228 and subsequent challenge infection.

a Experimental Setup: Vaccination of 12 hamsters with low-dose OTS-228 (10¹–10² TCID₅₀/animal). Six contact animals were co-housed one day post-vaccination to monitor potential transmission. b Antibody Response: Sera from 19 dpv were analyzed by RBD-specific ELISA. Six vaccinated hamsters showed a specific immune response (“responders”), while the others were designated “non-responders.” c Viral Genome Detection Post-Vaccination: Viral genome copies in nasal wash samples were quantified by RT-qPCR (orf1ab-specific), confirming virus presence in five of 12 vaccinated animals. d Survival Post-Challenge: Vaccinated animals were challenged with SARS-CoV-2 WT at day 21 post-vaccination. One responder died during sampling, while two non-responders succumbed to infection by day 7. e Body Weight Changes Post-Challenge: Responders maintained body weight, while non-responders exhibited significant weight loss by day 5 post-challenge. Statistical analysis was performed using a mixed-effects model with Geisser-Greenhouse correction and Sidak’s multiple comparisons test, with individual variances computed for each comparison between responder and non-responder groups. f Viral Shedding Post-Challenge: RT-qPCR analysis of nasal wash samples revealed significantly lower viral genome copies in responders compared to non-responders. Statistical analysis was performed using a mixed-effects model with Geisser-Greenhouse correction and Tukey´s multiple comparisons test, with individual variances computed for each comparison between responder and non-responder groups. g, h Viral Genome in Respiratory Organs: At 5 dpc, lung samples from non-responders contained high viral loads, while responders showed minimal virus. By 14 dpc, viral genomes were still detectable in non-responders but not in responders. i Histopathology, pneumonia-related atelectasis given in % affected area. Representative hematoxylin-eosin stained lung sections (bar 2.5 mm) are shown: (j) Lack of atelectasis in all responders, (k) moderate atelectasis (53%) in the non-responder at 5 dpc, (l) severe atelectasis (76–77%) in the two non-responding hamsters of 7/8 dpc and (m) lack of atelectasis for responders and (n) non-responders at 14 dpc. o Moderate hyperplasia and hypertrophy of type II pneumocytes (green arrow) for the non-responders at 14 dpc, bar 100 µm. p–r Antigen Detection: Immunohistochemistry revealed (q) minimal viral antigen in one of five responders (5 dpc) but (r) extensive antigen presence in three of six non-responders examined at 5/7/8 dpc, particularly in type I pneumocytes. s, t Seroconversion and Neutralizing Antibodies: All non-vaccinated hamsters seroconverted post-challenge, while neutralizing antibodies were present in responders, correlating with protection against lung infection. u Overall Conclusion: OTS-228 vaccination led to a robust humoral immune response in responders, correlating with protection against severe lung infection and virus replication. Non-responders experienced more severe outcomes, with higher viral loads and lung damage.